Spotlight pubs.acs.org/acschemicalbiology
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NEW METHOD FOR INTRODUCING SITE-SPECIFIC PROTEIN MODIFICATIONS
thus demonstrating the utility of their method for assessing the impact of post-translational modifications. Heidi Dahlmann
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Reprinted by permission from Macmillan Publishers Ltd: Nature Wolchok et al., 539, 443−447, copyright 2016.
Adapted from A. Yang, et al., Science 2016, 354, 623−626. Reprinted with permission from AAAS.
Immunotherapy, which takes advantage of an organism’s ability to target and destroy nonself cells or transformed/modified self such as certain tumor cells, is increasingly utilized as anticancer treatment. One type of immunotherapy, known as immune checkpoint blockade (ICB), involves administering antibodies that deactivate the checkpoint proteins that immune cells display in order to prevent hyperactivation and therefore protecting against autoimmunity. For example, recent clinical trials showed that ICB antibodies targeting the checkpoint ligands CTLA-4 and PD-1 produced long-lasting effects against various cancers. Unfortunately, some tumors are resistant to ICB therapy due to infiltration by tumor-associated myeloid cells (TAMCs), which suppress the immune response. Efforts to stamp out this form of ICB resistance were recently reported by a research team led by Jedd D. Wolchok and Taha Merghoub of the Memorial Sloan Kettering Cancer Center (Nature 2016, 539, 443−447). The team reasoned that administration of a pharmacological inhibitor of PI3Kγ, a protein highly expressed in TAMCs, would subvert TAMCmediated immunosuppression and make resistant tumors susceptible to ICB treatment. Indeed, treating model mice with the selective PI3Kγ inhibitor IPI-549 reduced the growth of resistant tumors. When given in combination with anti-CTLA-4 and antiPD-1 antibodies, IPI-549 significantly improved regression in certain model tumors compared with double checkpoint blockade treatment alone. Furthermore, mice in which tumors had been completely eliminated by combination therapy were resistant to tumor reimplantation, indicating the combination therapy had long-lasting effects. The efficacy of IPI-594 alone and in combination with PD-1 blockade in solid tumors is currently being tested in a phase 1 clinical trial.
The function of a protein is inextricably linked to its sequence of amino acid building blocks, the most common of which are the 20 naturally occurring amino acids encoded by the genome. Many of these amino acids can undergo post-translational modification such as alkylation or acylation of side chain functional groups. These modifications alter the hydrophobicity or charge of the amino acid residue, affecting the overall function of the corresponding protein. Scientists who wish to study the impact of site-specific posttranslational modifications have limited options for preparing modified proteins. Exposing a whole protein to an alkylating or acylating agent would indiscriminately label any or all reactive functional groups in the protein, so it is necessary to instead use chemical biology techniques like genetic code expansion, in which cellular translation machinery is reprogrammed to synthesize and incorporate aminoacyl tRNAs bearing modified amino acids. A research group led by Dieter Söll of Yale University and Hee-Youn Lee and Hee-Sung Park of the Korea Advanced Institute of Science and Technology has recently reported a new route to incorporating modified lysine residues that have eluded direct incorporation through genetic code expansion (Science 2016, 354, 623−626). The team exploited genetic code expansion to install O-phosphoserine (Sep) into specific positions in a variety of proteins. Treating the purified recombinant proteins with base led to the elimination of the phosphate group from the Sep side chains, generating enone products in their place. Metalpromoted conjugate addition of alkyl iodides into the enone products produced the desired modified amino acid side chains in the target proteins. In a proof-of-principle experiment, the authors synthesized histone H3 protein containing mono-, di-, or trimethyllysine at a biologically relevant position and measured their effects on nucleosome assembly and transcription, © 2016 American Chemical Society
TURNING OFF RESISTANCE TO ANTICANCER IMMUNOTHERAPY
Heidi Dahlmann Published: December 16, 2016 3230
DOI: 10.1021/acschembio.6b01082 ACS Chem. Biol. 2016, 11, 3230−3232
ACS Chemical Biology
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REVERSIBLE METHYLATION OF TRNA REGULATES TRANSLATION
Spotlight
A LOOK AT NON-ENZYMATIC RNA COPYING
Adapted with permission from Zhang et al., ACS Cent. Sci., DOI: 10.1021/acscentsci.6b00278. Copyright 2016 American Chemical Society.
Cell survival, growth, proliferation, and differentiation all depend on a cell’s ability to convert information embedded in the genetic code into a specific array of protein products. It is well-known that gene expression, the overall process of transcription of DNA into mRNA and the translation of mRNA into protein, can be turned on or silenced by reversible chemical modifications of DNA or mRNAs. Now tRNAs (tRNAs), which mediate transcription, can be added to this list, according to new results from a research team led by Tao Pan and Chuan He at the University of Chicago (Cell, 2016, 167, 816−828). The research team was interested in identifying the cellular targets of ALKBH1, a member of a family of dioxygenase enzymes known to perform oxidations on nucleic acid substrates. They used UV radiation to cross-link all proteins bound to RNA in HeLa cells and then used immunoprecipitation and gel electrophoresis to isolate and separate ALKBH1RNA complexes. They discovered that ALKBH1 primarily bound to tRNAs containing adenosine nucleotides methylated at the N1 position (m1A) and that ALKBH1 catalyzed the removal of the methyl group from m1A, including on the translation-initiating tRNA molecule bearing the amino acid methionine (tRNAiMet). Knowing that demethylation of m1A changes the stability and lifetime of tRNA in cells and that levels of tRNAiMet significantly impact translation initiation and cell proliferation, the research team investigated the effect of ALKBH1 in HeLa cells. They discovered that cells lacking ALKBH1 had higher levels of translation initiation and elongation than did wild-type cells, while cells in which ALKBH1 was upregulated in response to glucose deprivation had reduced levels of protein synthesis. The authors note that their discovery uncovers reversible chemical modification of tRNA as a new mechanism of controlling gene expression at the post-transcriptional stage.
Because of its unique ability to both encode genetic information and catalyze chemical reactions, RNA was perhaps the transcendent biopolymer linking the chemistry of the prebiotic soup to the biochemistry of early, simple life forms. This idea, known as the RNA world hypothesis, was bolstered by the discovery of natural ribozymes catalyzing many fundamental reactions in the cell, including peptidyl transfer within the ribosome or transesterification within the spliceosome. Researchers have also uncovered many other catalytic activities for RNA using in vitro evolution and selection for particular reactions. Among the most compelling evolved ribozymes are those with ligase or polymerase activity, since these demonstrate that a ribozyme can regenerate its own genetic information, reading and copying it by Watson−Crick base pairing. A central question, however, is, when the first polymers of RNA were copied nonenzymatically prior to the rise of ribozymes, was Watson−Crick base pairing a key feature for the incoming monomers? To address this question, Zhang et al. (ACS Cent. Sci. 2016, DOI: 10.1021/acscentsci.6b00278) turned to X-ray crystallography using double-stranded synthetic RNAs carrying LNA 5′ methyl-C overhangs, poised to pair with incoming guanosine monomers that would link with the 3′ end of the complementary RNA strand. For this study, the researchers synthesized a new guanosine monomer that structurally resembles an activated ribonucleotide, but with a C−P bond in place to prevent productive chemistry. A family of structures was solved with this novel ribonucleotide, termed PZG, crystallized with RNAs of varying 5′ overhang and RNA helix length. Zooming in on these high resolution structures uncovered Watson−Crick and Hoogsteen base pairing between the RNA’s terminal cytosine and the ribonucleotide’s base, G, along with several other noncanonical hydrogen bonding interactions. As the single stranded content of the RNA grew from one base to three, more noncanonical pairings were found. Interestingly, even with one monomer forming a canonical base pair with the template, the distance between the primer strand 3′ hydroxyl and the leaving group of the nucleotide are too far apart for chemistry to be possible, hinting at a necessity for catalysts to enter the playing field. This study shows how crystallography can peer into putative mechanisms in an RNA world prior to modern biology.
Heidi Dahlmann
Jason Underwood
Reprinted from Cell, 167, Fange Liu et al. ALKBH1-Mediated tRNA Demethylation Regulates Translation, 816−828. Copyright 2016, with permission from Elsevier.
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DOI: 10.1021/acschembio.6b01082 ACS Chem. Biol. 2016, 11, 3230−3232
ACS Chemical Biology
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Spotlight
TOOL FOR TRACKING BACTERIAL PROTEINS
Reprinted with permission from Ho and Tirrell, J. Am. Chem. Soc., 138, 15098−15101. Copyright 2016 American Chemical Society.
Scientists would like to have better ways to label and track the movement of proteins within bacteria. Now, a Caltech team has reported a general strategy for labeling bacterial proteins with small molecule fluorophores, and they demonstrate its use in live cells (Ho, S. H. and Tirrell, D. A. J. Am. Chem. Soc. 2016, 138, 15098−15101). The use of tags such as green fluorescent protein has revolutionized biologists’ ability to observe cells with optical microscopes. But these large fluorescent proteins can sometimes interfere with the delicate localization and regulatory processes in bacteria. So researchers would like to have other options for attaching glowing beacons to cellular proteins. Ho and Tirrell designed their labeling system based on the human N-myristoyltransferase (NMT) enzyme, which links fatty acids to an N-terminal protein sequence and only occurs naturally in eukaryotic cells. They paired the enzyme with fatty acid linkers that included an azido group along with a BODIPY fluorescent dye modified with a cyclic alkyne. With bacterial proteins engineered to include the NMT-targeting sequence, the enzyme appends the azido-fatty acid to the N-terminal glycine residue of the protein. The azide then undergoes an efficient cycloaddition with the alkyne to attach the fluorophore. To test the strategy, Ho and Tirrell studied two proteins involved in cellular chemotaxis (Tar and CheA) and two proteins that direct cell division (FtsZ and FtsA). After verifying that the strategy efficiently labeled the proteins in cell lysates and with fixed cells, the team moved on to test the labeling method in live cells with plasmids that could induce expression of the proteins. They observed that labeled Tar and CheA localized to the cellular poles and labeled FtsZ and FtzA moved to the division septa as expected. Control cells without induced protein expression did not glow brightly, even when treated with the fatty acid and the fluorescent dye. Though this technique would not work for proteins that use N-terminal localization signals, this approach suggests a relatively general approach for labeling prokaryotic proteins, It could be especially useful when larger fluorescent protein tags interfere with protein function. Sarah A. Webb
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DOI: 10.1021/acschembio.6b01082 ACS Chem. Biol. 2016, 11, 3230−3232
