Spotlight pubs.acs.org/acschemicalbiology
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CHAPEROME COMPLEXES INFLUENCE TUMOR SURVIVAL
CELLS LINE UP FOR WEIGH-IN
Reprinted by permission from Macmillan Publishers Ltd.: Nature Biotechnology Manalis et al., 34, 1052−1059, copyright 2016.
Reprinted by permission from Macmillan Publishers Ltd.: Nature, Chiosis et al., 538, 397−401, copyright 2016.
In the quest for developing new antibacterial or anticancer compounds, researchers benchmark their progress by determining the effects of their drug candidates on a general population of cellsfor example, by calculating the concentration of drug that kills 50% of cells. Population studies like these do not take into account the effect of rogue cells such as rapidly proliferating cancer cells or slowly growing bacterial cells, both of which may contribute to the development of drug resistance in a patient. Thus, technologies that allow researchers to quickly and comprehensively track the growth of each cell in a sample would help them to predict the effectiveness of pharmacological treatment. A new device enabling the high-throughput measurement of single-cell growth rates has been developed by a team of researchers led by Scott R. Manalis of MIT (Nature Biotechnol. 2016, 34, 1052−1059). The device contains a narrow channel that sends cells through a serial array of oscillating cantilever sensors, the resonance frequencies of which change in response to the buoyant mass of each cell passing through one at a time, with the cantilevers spaced far enough apart that the cells have time to grow or shrink in response to stimuli in the flow media. Knowing the exact time cells take to get from one cantilever to the next, the research team could track the mass of the exact same cell throughout its passage through the device, with masses being measured so precisely that it was possible to extrapolate the growth rates of each individual cell in a sample in less than half an hour. Using their device, Manalis and co-workers followed the growth rate of various eukaryotic and bacterial cell lines, identifying subpopulations of cells with unusual growth kinetics that could possibly have clinical significance.
When cells are under stress, a host of chaperone proteins and other folding enzymes that assist in restructuring damaged proteins and protein complexes, collectively referred to as the chaperome, help bring the cells back to homeostasis. A team of researchers led by Monica L. Guzman and Gabriela Chiosis recently discerned differences between chaperome complexes in normal cells and two distinct types of cancer cellsvariances that could be exploited for personalized cancer therapy (Nature 2016, 538, 397−401). The research team observed that when they isolated the most abundant chaperone protein in human cells, heat shock protein 90 (HSP90), from cultured noncancerous cells, HSP90 resolved mainly as a single species. In contrast, HSP90 resolved in complexes with other proteins when isolated from cancer cells; depending on the isoelectric points of these complexes, cancer cells could be categorized into two distinct groups (type 1 and type 2). The HSP90 inhibitor PU-H71 bound to type 1 HSP90 protein complexes, dubbed the epichaperome, more tightly than to type 2 HSP90 complexes or free HSP90. Furthermore, knockdown of other heat shock proteins or accessory protein components of the epichaperome were more fatal to type 1 cells than to type 2 cells. Considering the sensitivity of the epichaperome to inhibition and its necessity for the survival of type 1 cells, the research team reasoned that the epichaperome would be a possible target for cancer therapy, especially because type 1 cells were present in over half of surveyed cancer cell lines originating from various solid tumors, leukemias, and lymphomas. The higher the concentration of epichaperome in type 1 tumor cells, the more likely they were to undergo apoptosis upon treatment with PU-H71, prompting the authors to suggest that prospective patients be tested not only for epichaperome presence but also abundance when considering development of epichaperome therapy.
Heidi A. Dahlmann
Heidi A. Dahlmann
Published: November 18, 2016 © 2016 American Chemical Society
2941
DOI: 10.1021/acschembio.6b00969 ACS Chem. Biol. 2016, 11, 2941−2943
ACS Chemical Biology
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Spotlight
SONAR PINGS FOR RNA-BINDING PROTEINS
proteins or RNA metabolism, adding these new RNA-binding players and SONAR to look for more may help unlock new clues. Jason G. Underwood
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METHYLTRANSFERASES SEE THE LIGHT
Adapted with permission from Horning et al. J. Am. Chem. Soc., 138, 13335−13343. Copyright 2016 American Chemical Society.
Image courtesy of Peter Fowler.
Methylation of proteins, RNA, and DNA occurs in all kingdoms of life, and though methyl may seem like a minor posttranslational modification, it often exudes major effects on the cell. For example, in eukaryotes, histone proteins receive specific methylation marks to alter the chromatin state and gene expression. Sites in ribosomal and transfer RNAs receive base or ribose methylation marks which regulate folding, stability, and ultimately protein synthesis. The methyltransferases (MTs) catalyze methyl group installation, and their diversity reflects the wide range of biomolecules upon which they act. Some MTs have been characterized in depth using biochemistry, genetics, or structural biology, but many of the approximately 200 MTs encoded by the human genome still remain uncharacterized. Recently, Horning et al. (J. Am. Chem. Soc. 2016, DOI: 10.1021/jacs.6b07830) described a new chemical toolkit for studying MTs within a complex lysate of cellular proteins. In MT reactions, the methyl group comes from S-adenosylmethionine (SAM), so the researchers reasoned that photoreactive versions of the reaction product, S-adenosyl-homocysteine (SAH), would serve as useful probes. They tested several photoreactive groups installed on the N-6 position of adenosine, along with a second variable functionality, either a fluorophore for protein labeling or a biotin group for affinity purification. Using the UV cross-linking-fluorophore version of SAH with protein gels visualized putative MTs within lysates generated from several cancer cell lines. Those bands eliminated by excess unlabeled SAH competition were deemed specific. The same competition trick was also used to prioritize hits when the biotin version was deployed to affinity purify cross-linked MTs, drastically cutting down the quantitative mass spectrometry hit list from 350 proteins to just 45. Many peptide hits came from known MTs that methylate various classes of biomolecules, but many MTs with unknown function were identified as well. Finally, the researchers demonstrated that their cross-linking-fluorophore SAH can be used for competitive fluorescence polarization experiments aimed at identifying small fragment electrophiles to covalently modify the nicotinamide N-methyltransferase, human NNMT. A specific inhibitor
Once transcription generates a new RNA, a symphony of RNA-binding proteins (RBPs) escorts the RNA through steps such as processing, modification, transport, quality control, and translation. With all of these important functions, it is no wonder that RBP genes make up a significant portion of the genes in the human genome. Recent proteomic studies demonstrated that canonical predictions of RBPs based on primary sequence are coming up short, and unexpected proteins such as the glycolytic housekeeping protein, GAPDH, can possess RNA-binding activity. To go looking for new RBPs, Brannan et al. (Mol. Cell 2016, DOI: 10.1016/j.molcel.2016.09.003) embarked on a series of biochemical experiments to affinity purify 12 known human RBPs from cellular lysates by a rapid HaloTag methodology and then assay the copurified protein inventory. Proteins copurifying in an RNase-dependent manner constituted candidate RNA-binding proteins with those copurifying with more than five of their HaloTag-RBPs, termed super interactors. Inspired by this super interactor signal, the researchers postulated that existing protein−protein interaction data sets from RBP affinity purification lacking RNase should contain novel proteins not previously implicated in RNA binding. To query these massive mass spectrometry data sets, they built a classification algorithm termed SONAR which uses complementary data from multiple RBP purifications and multiple studies to hone in on the common interactor neighborhoods. This sensitive method identified a wealth of annotated RBPs but also unveiled hundreds of new candidate RNA-binding proteins in yeast, flies, and humans. Many are C2H2-zinc finger proteins and other proteins usually bundled into transcriptional regulation. Follow-up experiments using enhanced cross-linking-immunoprecipitation sequencing (eCLIP-seq) on four SONAR-identified candidates validated that all four are bona f ide RNA binding proteins, displaying binding events throughout the transcriptome. The interactions are specific with highly enriched sequence motifs and positional preference along a pre-mRNA emerging from the analysis. Together, these results indicate that the well has not run dry for new RNA-binding protein discovery. Given that many genetic diseases can be attributed to RNA-binding 2942
DOI: 10.1021/acschembio.6b00969 ACS Chem. Biol. 2016, 11, 2941−2943
ACS Chemical Biology
Spotlight
which does not react with other MTs was identified, showing the utility of this toolkit for both profiling MTs and screening for new inhibitors. Jason G. Underwood
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PROBING RIBOSOME CONSTRUCTION
Reprinted from Cell, 167, Kawashima, S. A., et al. Potent, Reversible, and Specific Chemical Inhibitors of Eukaryotic Ribosome Biogenesis, 512−524. Copyright 2016.
Though ribosomes assemble all cellular proteins, scientists know relatively little about how these protein factories are built. Now researchers have developed chemical inhibitors of ribosome biogenesis in fission yeast (Schizosaccharomyces pombe) and have used those tools to tease out some of the details of ribosome construction (Kawashima, S. A., et al., Cell, 2016, 167, 512−524). Ribosomes are a complex assembly of both nucleotides and proteins, made up of two primary subunits60S and 40S. Ribosomal DNA is first transcribed in the nucleolus to produce preribosomal subunits within the nucleus that are eventually transported to the cytoplasm and are organized into functional ribosomes. In this study, Kawashima et al. focused on inhibitors of Midasin (Mdn1), an AAA+ family ATPase known to be important for assembling the 60S subunit. In a screen of more than 10 000 compounds against strains of fission yeast that lack multidrug resistance, the team found a heterocyclic triazinoindole. Further experiments showed that Mdn1 was a likely target of this compound, ribozinoindole-1 (Rbin-1), and inhibited cell growth. Using a series of in vitro and in vivo assays, the researchers showed that cellular growth relies on four of the six ATPase active sites within Mdn1, and that Rbin-1 and related compounds can act as potent inhibitors of that activity. They then carried out a series of assays using fluorescently labeled proteins and enzyme activity to discover where and when these inhibitors shut down ribosomal assembly. They also showed that a single mutation within Mdn1 can protect against chemical inhibition in both biochemical and cell-based assays. Their resulting model proposes several functions for Mdn1 in ribosomal assembly. First, in the nucleolus, Mdn1 helps with the organization of a pre-60S particle and brings in a second AAA+ ATPase, Rix7, that facilitates the export of these structures to the nucleoplasm. Mdn1 is then needed to remove two other proteins to prepare the pre-60S particle for its move into the cytoplasm for final assembly. In addition to providing these new mechanistic details, the Rbin compounds are the first drug-like inhibitors of ribosome assembly. Therefore, these results could serve as a basis for developing other chemical inhibitors for this family of enzymes and help researchers examine ribosome assembly as a potential target for new therapeutic agents. Sarah A. Webb 2943
DOI: 10.1021/acschembio.6b00969 ACS Chem. Biol. 2016, 11, 2941−2943
