Spotlight pubs.acs.org/acschemicalbiology
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POLYMERASE TURNED REVERSE TRANSCRIPTASE The enzyme reverse transcriptase (RT) copies an RNA template to synthesize the complementary DNA during cellular replication of retroviruses. After their discovery, retroviral RTs became a workhorse of molecular biologists and paved the way for the RNA profiling, or transcriptomics, first by arrays and later by RNA-seq. In contrast to the DNA-dependent DNA polymerases critical for genomic replication, RTs are not as accurate or processive, owing to their more open conformation and lack of a proofreading exonuclease activity.
From Ellefson et al., Science 2016 352, 1590−1593, DOI: 10.1126/science.aaf5409. Reprinted with permission from AAAS, Copyright 2016. From Auer et al., Cell Syst. 2016, 2, 402−411, DOI: 10.1016/ j.cels.2016.05.006. Reprinted by permission from Elsevier, Copyright 2016.
Recently, Ellefson et al. (Science 2016, DOI: 10.1126/ science.aaf5409) demonstrated an in vitro evolution scheme to uncover mutations that turn a DNA-dependent DNA polymerase into an RT. Their scheme began with KOD, an Archaeal B-type polymerase often used in high-fidelity polymerase chain reaction (PCR) due to its robust proofreading activity. With a randomly mutagenized KOD library in hand, they employed a self-replication scheme in a compartmentalized manner similar to emulsion PCR. The method allowed up to 109 variants to be screened at once. With each round of the self-replication reaction, KOD polymerases that could copy through a stretch of RNA bases were selected. Stringency was controlled by the length of the RNA stretch and the evolutionary path of individual RT variants was traced with deep sequencing. After identifying KOD residues that were important for RT activity, a composite reverse transcription xenopolymerase, or RTX, was constructed for further experiments. They go on to show that RTX and its proofreading exonuclease activity afford 3−10-fold higher fidelity than the most commonly used RT from Maloney murine leukemia virus. The introduction of RTX could streamline workflows because it can function in both RT and PCR steps. The researchers also point out that their directed evolution scheme could also be adapted to select polymerases that copy modified nucleic acids.
In a recent study, Auer et al. (Cell Syst. 2016, 2, 402−411, DOI: 10.1016/j.cels.2016.05.006) developed a high-throughput screen to hunt for genes involved in cell stiffness. Using a prior assay from their laboratories as inspiration, the researchers immobilized a library of 3844 E. coli deletion mutants in 1% agarose media, depositing individual clones into microtiter plate wells. Parallel wells housed the same bacterial strains but growing in liquid media. Using simultaneous optical density growth measurements for both flavors of culture, researchers could gauge each mutant’s GRABS score, an acronym for general regulators affecting bacterial stiffness. Mutants displaying inhibited solid agarose culture constituted candidates for cell stiffness genes, yielding a negative GRABS score. They go on to show that the scores are highly correlated with microscopy-based estimations of Young’s modulus, a measurement of elasticity. Of the thousands of mutants screened, 46 candidate genes were identified which altered stiffness. Some expected gene families involved in cell wall biogenesis were identified, but a range of diverse intracellular processes were also implicated, such as replication or metabolism. Finally, the researchers demonstrated that their GRABS methodology can screen for stiffness changes during culture with an additional chemical component, opening up a new avenue for small-molecule screens using relatively simple lab equipment.
Jason G. Underwood
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SCREENING FOR STIFFNESS The bacterial cell wall provides structural integrity and protects from osmotic pressure. Peptidoglycan is a primary component of the cell wall, and antibiotics such as penicillin kill bacteria by meddling with the integrity of the wall. Little is known about the genes that modulate the overall stiffness of bacteria and their cell wall. © 2016 American Chemical Society
Jason G. Underwood Published: July 15, 2016 1770
DOI: 10.1021/acschembio.6b00585 ACS Chem. Biol. 2016, 11, 1770−1772
ACS Chemical Biology
Spotlight
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USING CYSTEINES TO EXPAND DRUGGABILITY Although small molecules are powerful tools for understanding and modulating protein function, just a small fraction of human proteins are matched with small-molecule ligands. One approach for tackling this problem has been fragment-based ligand discovery (FBLD), a process of screening purified proteins against libraries of compounds, usually for hits that bind noncovalently. Now Backus et al. report a covalent FBLD strategy that takes advantage of endogenous cysteine residues within proteins to screen a library of small molecules against whole cells or cell lysates. They then use their ligand discovery strategy to probe caspase function in apoptosis (Nature, 2016, 534, 570−574).
most potent fragment to construct inhibitors of caspase-8 and caspase-10 and study their biochemical activity. This study shows the potential of combining FBLD and chemical proteomics to look for new drug targets, and the researchers expect that the strategy could also be employed to screen using other nucleophilic amino acid side chains. Sarah A. Webb
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DESIGNING AN ON-SWITCH FOR MEMBRANE PROTEINS Protein designers would like to construct novel structures that they can activate within living cells. But such proteins are so complex that it has been difficult to build switches that can turn on protein function. However, Kiyonaka et al. have now built a membrane protein that they can activate allosterically using a metal complex (Nat. Chem. 2016, DOI: 10.1038/nchem.2554).
From Kiyonaka, S. et al., Nat. Chem. 2016 DOI: 10.1038/ nchem.2554. Reprinted by permission from Macmillan Publishers Ltd., Copyright 2016
Though membrane proteins serve as cornerstones of cellular function, their complexity makes them especially difficult scaffolds for protein design. By building on earlier examples that used coordination chemistry to activate proteins, Kiyonaka et al. wanted to program in a new allosteric regulatory site in a membranebound glutamate receptor. This novel regulatory site would consist of two histidine residues. When these two side chains bound to an appropriate metal, they would snap shut like a venus fly trap, stabilizing the active conformation of the protein. First the team modified an AMPA receptor, creating a library of 8 proteins with histidine double mutants that could interact with chelating metals. They then transfected cells with these mutant proteins and used a fluorescent calcium screening technique to assess the activity of the proteins and their response in the presence of zinc, nickel, and palladium ions or their chelating complexes. Pd(bpy) successfully activated two of the eight mutants. Using NMR and titration studies, the team showed that Pd(bpy) is essential for activity along with glutamate. The new protein is also more sensitive to glutamate than the wild-type receptor. The team also tried the same strategy with the GPCR-type mGluR1 receptor, but in that case, the allosteric interaction activated the protein on its own, even in the absence of glutamate. Finally, the team demonstrated that they could activate their AMPA receptor mutant in cultured neurons. This novel design
From Backus, K.M., et al., Nature, 2016, 534, 570−574. Reprinted by permission from Macmillan Publishers Ltd., Copyright 2016.
The strategy modifies a method of studying cysteine reactivity using proteomics. Whole cells or lysates were exposed either to a library of fragments that contained electrophiles known to react with cysteines or to DMSO. These samples were then modified with a light isotope tag for the library fraction and a heavy isotope tag with the DMSO fraction. Ultimately the fractions were pooled, digested, and analyzed via LC-MS. Using sophisticated quantitative analysis to compare the fragments, they found more than 750 liganded cysteines on nearly 650 liganded proteins out of a total 6150 cysteines on 2900 proteins. Only a subset of these proteins have known small-molecule probesjust 14% of these proteins were also found in the DrugBank database. The team observed that several of the small-molecule fragments targeted the catalytic cysteine residue in caspase-8, a protease whose role in apoptotic signaling is not well understood. Surprisingly the probes selectively labeled the inactive proenzyme rather than its activated form. The team performed further chemical studies and optimized the structure of the 1771
DOI: 10.1021/acschembio.6b00585 ACS Chem. Biol. 2016, 11, 1770−1772
ACS Chemical Biology
Spotlight
Patients with irritable bowel syndrome can develop abdominal pain on the basis of mechanical sensitivity. In ex vivo studies and using mouse colonic tissue, the team showed that Hm1a increased the sensitivity of mechanical responses, which could be inhibited with a broader-spectrum Nav channel inhibitor. This approach could provide novel therapies for irritable bowel syndrome and other conditions mediated by this pathway.
strategy could be applicable in other neurotransmitter receptors and could provide new ways to modulate the function of a range of membrane proteins. Sarah A. Webb
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SPIDER TOXINS REVEAL PAIN MECHANISM Voltage-gated sodium channels kick off the action potentials in most neurons, including within the nerve fibers that signal various type of pain. However, researchers have had limited tools to study how different channel subtypes might signal chemical, thermal, or mechanical pain. Now an international team of researchers has discovered a set of spider toxins that selectively interact with one type of channel, Nav1.1, and have shown that it regulates nerve fibers that sense mechanical pain (Nature, 2016, 534, 494−499, DOI: 10.1038/nature17976).
Sarah A. Webb
From Osteen, J. D. et al., Nature, 2016, 534, 494−499, DOI: 10.1038/nature17976. Reprinted by permission from Macmillan Publishers Ltd., Copyright 2016.
First Osteen et al. screened dozens of toxins and isolated two active peptides from tarantula (Heteroscodra maculata) venom, which they named Hm1a and Hm1b. Then they showed that Hm1a selectively activated Nav1.1 channels by inhibiting channel activation, thereby boosting the spike frequency and lengthening the action potential. The activity of Hm1a suggested that it bound to the S3b-S4 voltage sensor region of the protein, and the team constructed protein chimeras to show that the S1−S2 loop of the Nav1.1 protein was also critical for Hm1a activity. The identification of this new loop could suggest a strategy for finding ways to target other subtypes of Nav channels. Using immunohistochemistry, the team showed that Nav1.1 channels occur on myelinated Aδ sensory neurons. In ex vivo experiments, Hm1a boosted the firing rate in Aδ fibers that respond to mechanical pain, suggesting that Nav1.1 played a role in that process. Finally, the team studied Nav1.1’s role in pain sensation using knockout mice and Hm1a, showing that the mechanical pain behavioral responses were indeed triggered by the toxins in an Nav1.1-dependent manner. 1772
DOI: 10.1021/acschembio.6b00585 ACS Chem. Biol. 2016, 11, 1770−1772
