Spotlight pubs.acs.org/acschemicalbiology
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ASTRIN BUFFERS MTOR ACTIVITY IN STRESSED CELLS
These activities occur when a cell is not dividing. Therefore astrin has dual and exclusive roles in mitosis and in regulating mTOR. In healthy cells, astrin may serve as a safety switch that prevents cells from self-destructing during minor, short-lived stresses. This protein is highly expressed in cancer cells, however, which suggests that this antiapoptotic protein may be an important target for new cancer drugs. Sarah A. Webb, Ph.D.
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MANIPULATING ENVIRONMENTAL STRESS
Okamoto, M., et al., Proc. Natl. Acad. Sci. U.S.A., 110, 12132− 12137. Copyright 2013 National Academy of Sciences, U.S.A. Reprinted from Cell, 154, Thedieck, K., et al., Inhibition of mTORC1 by Astrin and Stress Granules Prevents Apoptosis in Cancer Cells, 859−874. Copyright 2013, with permission from Elsevier, Inc.
Abscisic acid (ABA) is a plant hormone involved in the response to environmental stress, such as drought or excessive heat. Receptors for ABA, part of the START superfamily of ligandbinding proteins, are part of an intricate signaling network conserved across land plants that regulates the stress response. The mechanisms by which ABA receptors function, however, are not well-defined, especially given that some are dimers in solution while others are monomeric. Now, Okamoto et al. (Proc. Natl. Acad. Sci. U.S.A., 2013, 110, 12132−12137) report the discovery of the dyhydroquinolinone sulfonamide quinabactin, a novel ABA agonist and valuable probe of ABA receptor signaling in plants. Quinabactin was discovered using a yeast two-hybrid reporter gene assay in which approximately 57,000 compounds were screened for their ability to induce ABA receptor activity. Like ABA, quinabactin suppresses water loss and promotes drought tolerance in adult Arabidopsis and soybean plants. Using various genetic analysis methods including transcriptional analysis in Arabidopsis mutants, microarrays, and quantitative RT-PCR, the authors demonstrate that quinabactin induces similar transcriptional responses as ABA, rescues multiple phenotypes in ABAdeficient plants, and activates dimeric ABA receptors. Analysis of the X-ray structure of quinabactin bound to the dimeric receptors points to its participation in a key hydrogen bonding network, and this interaction likely influences its biological activity. The identification of this synthetic ABA agonist enables chemical control of ABA receptor activity and presents an opportunity to manipulate the response of plants to environmental conditions.
A central hub for growth and metabolism in cells, the mTOR (mammalian target of rapamycin) kinase serves as a sensor switch to respond to environmental cues. Under oxidative stress, the mTORC1 complex prompts cells to produce stress proteins. If left unchecked, this process spurs the cell into hyperactivity and leads to apoptosis. Now Thedieck et al. (Cell, 2013, 154, 859−874) have found a molecular off-switch for this process in cancer cells. Under duress, cells bundle proteins and mRNA into cytosolic packages called stress granules. These structures can inhibit apoptosis and can be sites for translation in stressed cells. Recent research had suggested that these structures played an unexplained role in inhibiting mTOR. To find the molecular culprit, the researchers looked for proteins that immunoprecipitated with mTOR and its binding proteins raptor and rictor. Astrin, a protein associated with mitosis, bound to raptor but not rictor or mTOR, which suggested that it bound to this protein when it was not part of an mTORC1 complex. Thedieck et al performed a series of experiments to better understand these interactions within cancer cells. They showed that astrin can suppress signaling from the mTORC1 complex. In addition, fluorescence imaging showed that astrin binds to stress granules and can sequester raptor on those structures. In the presence of oxidants, astrin prevented raptor and mTOR from associating, and kept mTOR from stimulating apoptosis in response to stress. © 2013 American Chemical Society
Eva J. Gordon, Ph.D. Published: September 20, 2013 1857
dx.doi.org/10.1021/cb4006497 | ACS Chem. Biol. 2013, 8, 1857−1859
ACS Chemical Biology
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MINDING OUR QS FOR TUBERCULOSIS
Spotlight
CHECKING OUT THE ATM CHECKPOINT
Reprinted by permission from Macmillan Publishers Ltd: Nat. Med., advance online publication, 4 August 2013, DOI: 10:1038/nm.3262.
In 2011, close to 9 million people worldwide contracted tuberculosis, and approximately 1.4 million died from the disease. Nearly 4% of tuberculosis cases are multidrug-resistant strains, stressing the urgent need for new drugs that work by novel mechanisms of action. To this end, Pethe et al. (Nat. Med. advance online publication August 4, 2013; DOI: 10:1038/nm.3262) report the discovery of Q203, a new clinical candidate for the treatment of tuberculosis. Q203 was discovered by screening over 120,000 small molecules in a phenotypic high-content screen. In the screen, macrophages infected with a fluorescent strain of Mycobacterium tuberculosis, the causative agent of tuberculosis, were treated with the compounds, and the bacterial load and macrophage number in response to compound exposure was assessed. Importantly, Q203 was also effective against drugresistant strains of M. tuberculosis. In mouse models of both acute and established tuberculosis, treatment with Q203 led to at least 90% reduction in bacterial load. Notably, toxicity and pharmacokinetic studies in vitro and in rodents suggest that Q203 is safe and well-tolerated. Whole-genome sequencing of mutants spontaneously resistant to Q203 led to the identification of the respiratory cytochrome bc1 complex as the molecular target of Q203, and also pointed to the key role of a threonine residue in the ubiquinol-binding active site of the complex. The involvement of cytochrome bc1 suggests that the compound works by interfering with ATP synthesis, and structural analysis of cytochrome bc 1 complexes from other organisms offered additional insight into the interaction between the cytochrome bc1 and Q203. Together, the results introduce a promising new lead for tuberculosis therapy and support targeting ATP synthesis as valid strategy for tuberculosis drug discovery efforts.
Shen, C. and Houghton, P. J., Proc. Natl. Acad. Sci. U.S.A., 110, 11869−11874. Copyright 2013 National Academy of Sciences, U.S.A.
Eva J. Gordon, Ph.D.
Eva J. Gordon, Ph.D.
The ataxia telangiectasia mutated (ATM) checkpoint is a guardian for the genome, helping to ensure appropriate cellular responses to DNA damage such as cell cycle arrest, DNA damage repair, or cell death. Suppression of this checkpoint enables the perseverance of DNA mutations, a mechanism that unfortunately is exploited in some childhood cancers. Toward thwarting this mechanism, Shen and Houghton (Proc. Natl. Acad. Sci. U.S.A., 2013, 110, 11869−11874) explore the molecular underpinnings of ATM suppression in cellular and animal models of childhood sarcoma. Upregulation of mammalian target of rapamycin (mTOR) signaling is common in many cancers; the authors hypothesized that the lower levels of ATM mRNA (mRNA) and protein observed in tissue derived from childhood solid tumors might be linked to mTOR signaling gone awry. Indeed, in both cell lines and mouse xenograft models for rhabdomyosarcoma (a common childhood cancer that arises from skeletal muscle precursor cells), inhibiting the mTOR pathway led to an increase in ATM mRNA and protein levels. This suggests that mTOR signaling suppresses ATM in cells. Investigation of the mechanism for this suppression pointed to the downstream target of mTOR, S6K, that positively regulates a transcription factor, MYCN. Upregulation of MYCN was found to positively regulate the expression of two microRNAs (miRNAs) that suppress expression of ATM, and downregulation of these miRNAs contributes to elevated ATM. These findings illuminate a path by which tumor cells cultivate genomic instability, and suggest inhibition of mTOR activity as a potential therapeutic strategy for cancers characterized by compromised ATM function.
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dx.doi.org/10.1021/cb4006497 | ACS Chem. Biol. 2013, 8, 1857−1859
ACS Chemical Biology
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Spotlight
I’LL BE BACK FOR NEW TERMINATORS
Reprinted by permission from Macmillan Publishers Ltd: Nat. Methods, Chen, Y. J., et al., 10, 659−664, copyright 2013.
In bacteria, transcription termination takes place when the RNA polymerase synthesizes a special termination signal consisting an RNA hairpin followed by a U-rich sequence. This pair of features, shared by many known terminators is often used to search genome space for putative new terminators. These sequences are also valuable to those who engineer recombinant bacterial strains since a single promoter can drive the expression of multiple encoded genes, with terminator sequences added to tune the levels of each component. As genetically engineered circuits become more complicated with dozens of genes in a construct, an abundance of highly similar terminator sequences can be problematic. The homologous recombination machinery can edit out the terminators since they appear to be a sea of duplications. In the hunt for a more diverse set of terminator sequences, Chen et al. (Nat. Methods 2013, 10, 659−664) screened 317 natural terminators in a clever cell-sorting assay. One promoter drove the expression of both green and red fluorescent proteins, with a terminator sequence of interest between the two genes. This allowed calculation of terminator strength from the ratio of the green and red fluorescence intensities. Of the natural terminators, 87 showed termination efficiencies greater than 90%. Armed with a rich set of data, the researchers compared features of the most efficient terminators to learn what biophysical principles might govern their strength. The U-rich tract was extremely important, but it appeared that overall hairpin strength was not as important as kinetically fast-forming hairpins, such as those closed by tetraloops. After noting a few of these design tips, the researchers set out to create synthetic terminators that were not previously observed in biological systems. The final result was a set of 265 new synthetic terminators, with almost one-third of them displaying a termination efficiency greater than 90%. Most interestingly, the new synthetic terminators had less than 25 base pairs of contiguous identical sequence with one another, a critical feature for avoiding homologous recombination. This study gives a new look into features that make RNA polymerase fall off at a terminator and it gives the genetic engineers a bigger toolbox for stitching together even more complicated gene expression circuits. Jason G. Underwood, Ph.D.
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dx.doi.org/10.1021/cb4006497 | ACS Chem. Biol. 2013, 8, 1857−1859
