Spotlight pubs.acs.org/acschemicalbiology
O
rapid resolution. It is known that to resolve the branch site and liberate the spliced exteins from the intein, an asparagine side chain performs a nucleophilic attack on the adjacent peptide bond. The mystery of the intein reaction lies at that asparagine, an amino acid that rarely act as a nucleophile. To better understand the mechanism, Liu et al. (Proc. Natl. Acad. Sci. U.S.A. 2014, DOI: 10.1073/pnas.1402942111) constructed a version of the well-studied Mycobacterium DNA gyrase A intein via a semisynthetic approach. Because a portion of the protein was produced by solid-phase peptide synthesis, a wide array of unnatural side chains could be substituted. Substituting an intein’s natural histidine which is key for debranching to a β-thienyl-alanine afforded the steric qualities of histidine but would not support catalysis. With this trapped intermediate construct in hand, the researchers turned to X-ray crystallography to get a better look at the reaction center. What they found was a surprising conformation for the asparagine side chain in question. It displays a nearly optimal angle of approach for nucleophilic attack on the amide backbone and is stabilized in this position by a hydrogen bond between the asparagine’s side chain oxygen and the backbone amide bond of a nearby valine residue. The semisynthetic approach then allowed atomic mutagenesis of this site, replacing the amide bond with an ester, a substitution that was completely inactive for branch resolution, strongly supporting a role for the hydrogen bond in activation of the nucleophile. While inteins were first discovered more than 25 years ago, this study unlocks a mystery and gives new insight into the catalytic capabilities of asparagine residues. Jason G. Underwood, Ph.D.
ur editors have picked their favorite chemical biology papers published in 2014. The list features content from ACS Chemical Biology as well as other journals. We hope you enjoy the selections. Wishing all our readers a Happy New Year!
■
THE BATTLE FOR BTK INHIBITORS
Reprinted with permission from Wu et al. ACS Chem. Biol., 9(5), 1086−1091. Copyright 2014 American Chemical Society.
Members of the Tec family of nonreceptor tyrosine kinases are key regulators of the growth and differentiation of blood cells. However, deregulation of the Tec kinase BTK has recently been linked to several blood cell cancers including certain leukemias and lymphomas, spurring an intense hunt for BTK inhibitors with therapeutic activity. Adding to the small arsenal of reversible and irreversible BTK inhibitors currently being evaluated in both preclinical and clinical studies, Wu et al. (ACS Chem. Biol. 2014, DOI: 10.1021/cb4008524) now report the discovery of a novel, potent, and selective covalent inhibitor of Btk. Strategically combining structure-based drug design, kinome profiling, and cellular assays, the authors discover QL47, a tricyclic quinolone-based BTK inhibitor. QL47 covalently binds to a cysteine residue on Btk, inhibiting its kinase activity. Interestingly, the compound also promotes degradation of Btk. This dual action may contribute to its increased potency relative to other Btk inhibitors in preventing the growth of Bcell derived cancer cells. Eva J. Gordon, Ph.D.
■
■
ALLOSTERY AS THERAPY
REVEALING THE INTEIN’S SECRET Reprinted with permission from Cho et al., ACS Chem. Biol., 9, pp 2334−2346. Copyright 2014 American Chemical Society.
Schizophrenia is a complex psychiatric disorder, but in recent years, a number of genomics approaches have uncovered a set of risk genes. Among the hits is the metabotropic glutamate receptor, mGlu1, which harbors rare nonsynonymous variants in schizophrenics. To characterize the activity of these mutant receptors, Cho et al. (ACS Chem. Biol. 2014, DOI: 10.1021/ cb500560h) assayed nine stable cell lines that expressed various versions found in patients. The results demonstrate that mutant mGlu1 receptors display lower activity in calcium signaling, but not due to mislocalization. To attempt to rescue the mutant receptor phenotype, a family of positive allosteric modulators, or PAMs,
Liu et al., Proc. Natl. Acad. Sci. U.S.A., DOI: 10.1073/pnas.1402942111. Copyright 2014 National Academy of Sciences, U.S.A.
Characterizing a branched intermediate was key to understanding the first chemical step of RNA splicing, but getting a look at the transient branched species found in the self-splicing of proteins known as inteins has remained elusive due to its © 2015 American Chemical Society
Published: January 16, 2015 7
DOI: 10.1021/acschembio.5b00012 ACS Chem. Biol. 2015, 10, 7−11
ACS Chemical Biology
Spotlight
various diseases related to aging or protein misfolding. Eva J. Gordon, Ph.D.
were synthesized with inspiration from small molecules that act allosterically on the related mGlu4 receptor. Two of the PAMs show promise in partially restoring the signaling activity of several mutant mGlu1 receptors. Finally, animal studies lead the researchers to postulate that the heterogeneous nature of schizophrenia will likely require a range of therapeutic options depending on the specific mutations in each patient. Jason G. Underwood, Ph.D.
■
■
LABELING MEMBRANES PROTEINS IN A SNAP
HARNESSING THE HEXOSAMINE PATHWAY
Reprinted with permission from Prifti, E. ACS Chem. Biol. 9, 606−612, Copyright 2014 American Chemical Society.
Labeling proteins with fluorescent probes is an incredibly powerful and important approach for investigating biological processes. Nile Red is a dye whose fluorescence depends on the polarity of the environment; the dye is highly fluorescent in apolar environments such as cell membranes but practically invisible in polar environments such as water. Prifti et al. (ACS Chem. Biol. 2014, DOI: 10.1021/cb400819c) exploit this unique property of Nile Red to develop a method for labeling membrane proteins. Integral to their approach is the use of SNAP-tag, an enzyme that reacts specifically with O6-benzylguanine moieties. The authors synthesize O6-benzylguanine-containing Nile Red derivatives and demonstrate their utility for labeling various SNAP-tagged membrane proteins. Notably, this strategy enables imaging of membrane proteins without the requirement for a washing step, simplifying the experimental process and enabling real-time monitoring of membrane protein activity. This study presents an exciting new tool for investigating this notoriously challenging group of proteins. Eva J. Gordon, Ph.D.
Reprinted from Cell, 156, Denzel, M. S., et al., Hexosamine Pathway Metabolites Enhance Protein Quality Control and Prolong Life, 1167−1178, copyright 2014, with permission from Elsevier.
Aging, the extraordinarily complex but inevitable process that so many seek to elude, devolves through an accumulated decline in function at the cellular, organ, and whole organism level. Protein quality control systems, which regulate protein synthesis, folding, maturation, and removal in the cell, play a key role in longevity, and misregulation in these systems is associated with a variety of age-related diseases. The nematode Caenorhabditis elegans has been a pioneering model system to study the influence of protein quality control on longevity, as exemplified by numerous long-lived mutant strains that are able to sustain protein homeostasis to older ages. Exploring this remarkable phenomenon further, Denzel et al. (Cell, 2014, 156, 1167−1178) discover that certain mutations in an enzyme called GFAT-1 lead to diminished susceptibility to aging disorders and increased lifespan. The authors developed a screen to identify C. elegans mutants resistant to treatment with tunicamycin, an inhibitor of the synthesis of N-glycans, which are essential for the proper maturation and folding of many secreted proteins. Certain mutations that activate GFAT-1, a key enzyme in the hexosamine pathway responsible for synthesizing precursors for N-linked and O-linked glycans, conferred resistance to tunicamycin. Activation of the hexosamine pathway, as well as supplementation with the N-glycan precursor N-acetylglucsoamine, led to lifespan extension and protection from certain age-related neurotoxicities. Though an association between protein quality control and longevity is well established, this study uncovers an unexpected link between hexosamine metabolites and the protein quality control system that was previously unappreciated. These findings suggest that enhancing the capacity of the cell’s protein quality control mechanisms through hexosamine pathway activation or supplementation with N-acetylglucosamine may be a therapeutic strategy for
■
ADDING NEW BASES TO THE DOUBLE HELIX
Reprinted by permission from Macmillan Publishers Ltd.: Malyshev et al. Nature 2014, 509, 385−388.
Accurate replication of DNA by polymerases is essential for life, so it is no surprise that these enzymes have evolved such high fidelity. Forming additional layers of quality control for the genome, polymerases often proofread their own work while DNA repair enzymes can detect and fix damaged sites. In a test tube, researchers have used polymerases to add new base pair combinations to the natural G:C and A:T double helix, but could unnatural nucleotides stand a chance at replication 8
DOI: 10.1021/acschembio.5b00012 ACS Chem. Biol. 2015, 10, 7−11
ACS Chemical Biology
Spotlight
biological and therapeutic applications. Eva J. Gordon, Ph.D.
among the rigorous scrutiny of a living cell’s machinery? Now, Malyshev et al. (Nature 2014, 509, 385−388) have answered that question by engineering an E. coli strain that can replicate with six letters of genetic code. The additional base pair was made up of two hydrophobic bases, d5SICS and dNaM, which form complementary interactions without the presence of hydrogen bonds. The new base pair was inserted into a plasmid set up for replication by the bacteria’s DNA polymerase I. Because cells cannot synthesize nucleoside triphosphates for incorporating unnatural bases, these were provided exogenously in the medium and a diatom gene, nucleoside triphosphate transporter 2, NTT2, was expressed to bring these into the cell. Because the transporter was linked to an inducible promoter, the researchers could turn on and off the cell’s access to the unnatural dNTPs, providing an array of convincing controls. With the transporter on and the d5SICS and dNaM triphosphates in the media, recovered plasmids harbored the unnatural bases as detected by mass spectrometry of a complete plasmid digest and by abrupt termination at the site during Sanger sequencing. Even in stationery growth experiments, the plasmid maintained the unnatural base pair, indicating that it is evading the DNA repair machinery. These experiments offer an exciting path forward where an expanded genetic code could extend into transcription and translation to offer up a whole new set of functional groups to the cell’s repertoire. Jason G. Underwood, Ph.D.
■
■
FATTENING UP ON CELL DIVISION
Reprinted from Cell, Atilla-Gokcumen, G.E.; et al., Dividing Cells Regulate Their Lipid Composition and Localization, January 23, 2014, DOI: 10.1016/j.cell.2013.12.015, with permission from Elsevier.
Lipids perform numerous functions in the cell, ranging from their involvement in signaling networks to providing mechanical support for the inherently curved architecture of the cell membrane. Surprisingly little, however, is known about the role of lipids in the fundamental process of cell division. Now, Atilla-Gokcumen et al. (Cell 2014, DOI: 10.1016/ j.cell.2013.12.015) conduct a comprehensive and quantitative analysis of lipid composition and localization in dividing cells. The authors use a variety of techniques to investigate the role of lipids in cell division. Liquid chromatography−mass spectrometry (LC-MS) was employed first to profile the lipidome (encompassing the thousands of lipid species present in the cell) in cells synchronized at different stages of the cell cycle. Compared with cells in S phase, dividing cells exhibited increased levels of 11 lipid species, some of which are expressed at very low levels in nondividing cells and whose biological roles are poorly defined. Atomic force microscopy was used next to probe the mechanical properties of the cell membrane at various stages of the cell cycle. The membrane was found to withstand higher force during cell division, and the different mechanical properties could be attributed, at least in part, to the physical properties of the distinct lipid composition of the dividing cell membranes. Finally, RNAi was used to perturb lipid levels in cells, specifically via knockdown of enzymes involved in lipid biosynthesis. Strikingly, 23 lipid biosynthesis genes were implicated in cell division. These findings support a hypothesis in which specific lipids and lipid families likely play important structural and signaling roles in the cell division process, and indicate that the cell has regulatory processes directing lipid biosynthesis and localization as it moves through its life cycle. Eva J. Gordon, Ph.D.
PUTTING A FINGER ON PROTEIN DELIVERY
Reprinted with permission from Gaj et al., ACS Chem. Biol., 9, 1662−1667. Copyright 2014 American Chemical Society.
As the field of protein-based therapeutics continues to expand, the ability to efficiently deliver proteins into cells is increasingly important. Current methods, such as cell-penetrating peptides, liposomes, and virus-like particles, are limited by various factors including poor efficiency, cytotoxicity, and diminished protein activity. Now, Gaj et al. (ACS Chem. Biol. 2014, DOI: 10.1021/ cb500282g) exploit the inherent cell-permeability of zinc-finger nuclease proteins through the fabrication of efficient zincfinger-based protein delivery machines. The authors demonstrate that fusion of two or three zinc finger domains to a green-fluorescent protein variant or firefly luciferase facilitates highly efficient delivery into numerous normal and cancerous cell lines, without compromising the protein’s fluorescence or enzymatic activity, respectively. Probing the mechanism of zinc-finger-mediated delivery, they determine that the fusion proteins enter the cell through macropinocytosis. Further development of zinc-finger domains as protein delivery agents has exciting potential for numerous
■
BETTING ON KINASE INHIBITORS
Reprinted with permission from Ember et al. ACS Chem. Biol., 9, 1160−1171. Copyright 2014 American Chemical Society.
9
DOI: 10.1021/acschembio.5b00012 ACS Chem. Biol. 2015, 10, 7−11
ACS Chemical Biology
Spotlight
provides a blueprint for creating fluorogenic folding probes, probes that can be used in a plate reader to quantify the folded fraction of a protein-of-interest to explore how protein folding dynamics influence cellular function. Eva J. Gordon, Ph.D.
Bromodomain-containing proteins, such as those in the bromodomain and extra terminal (BET) family, bind acetylated lysine residues on histones. This integral role in epigenetic regulation has made them intriguing drug targets for diseases influenced by epigenetic changes in the cell, such as certain cancers and inflammatory conditions. Inspired by the recent discovery that a cyclin-dependent kinase inhibitor also bound to the acetylated lysine recognition site of a BET protein, Ember et al. (ACS Chem. Biol. 2014, DOI: 10.1021/cb500072z) explore whether other kinase inhibitors might also interact with the BET protein BRD4. The authors employ a robotic cocrystallization process to determine the structures of BRD4 crystals that grow in the presence of various kinase inhibitors. Of 581 inhibitors screened, 14 were identified as binders of the acetylated lysine site. These findings suggest that BET proteins may be offtargets of kinase inhibitors. This opens the door to the development of novel BET inhibitors as well as dual BETkinase inhibitors and may offer insight into the development of more selective kinase inhibitors with reduced off-target effects. Eva J. Gordon, Ph.D.
■
■
NEW WEAPONS AGAINST EBOLA
Reprinted with permission from Chen et al. ACS Chem. Biol., 9, 2263−2273. Copyright 2014 American Chemical Society.
A surge in Ebola infections in Africa has researchers working around the clock to identify a possible therapy for this often deadly disease. Now, a new study puts the Sudan strain known as SUDV in the crosshairs while hunting for neutralizing antibodies. Using a library of humanized antibody candidates coupled with a phage display method, Chen et al. (ACS Chem. Biol. 2014, DOI: 10.1021/cb5006454) uncovered a family of 17 synthetic antibodies that bind specifically to the SUDV envelope glycoprotein. Further studies with live virus in tissue culture and mice narrowed the field to two strong therapeutic candidates. With either of these antibodies in their bloodstream, young mice were protected from a lethal challenge of SUDV and even exhibited memory immunity when survivors were tested with a rechallenge. The results are promising and begs the question; could these antibodies or others be recruited to the fight against this deadly virus? Jason G. Underwood, Ph.D.
PROBING PROTEIN FOLDING IN CELLS
■
Liu, Y., et al., Proc. Natl. Acad. Sci. U.S.A., 2014, 111, 4449−4454 DOI: 10.1073/pnas.132368111. Copyright 2014 National Academy of Sciences, U.S.A.
NONRIBOSOMAL PEPTIDE SYNTHETASES: NOW ACCEPTING CLICKABLE SUBSTRATES Proteins, nucleic acids, carbohydratesthese are just a few of the very large biomolecules assembled by protein complexes in the cell. Smaller natural products such as polyketides and peptides are also constructed from monomeric building blocks by dedicated assembly line-like complexes of proteins called synthetases. Bacterial nonribosomal peptide synthatases (NRPSs), for example, produce the peptide antiobiotics gramacidin S and tyrocidine. To study the activities or to improve the therapeutic potential of natural products, chemical biologists often chemoselectively label the molecule of interest by attaching alkyne or azide functional groups, which can undergo bioorthogonal “click” reactions to covalently tether it to target substrates or biomacromolecules. Chemists can use solid-phase protein synthesis to insert clickable amino acids into peptides; however, solid-phase synthesis cannot mimic the ability of NRPSs to catalyze epimerizations or other modifications necessary for producing specific natural products. Therefore, a team of researchers led by Donald Hilvert at the ETH Zurich sought to engineer NRPSs themselves to produce labeled natural products biosynthetically (Angew. Chem. Int. Ed., 2014, DOI: 10.1002/anie.201405281). In an NRPS, modules called adenylation (A) domains select specific amino acids for incorporation into the peptide product. Thus, Hilvert and co-workers focused on mutating A domains, in this case derived from the gramicidin S and tyrocidine NRPSs, so that these domains would accept alkyne- and azidecontaining amino acids. The mutant A domains processed the
Though strung together as a linear collection of amino acids, proteins must fold into intricate three-dimensional structures in order to function properly. Through this process, various states of properly folded, misfolded, and aggregated proteins can coexist. It is relatively simple to distinguish aggregated, insoluble protein from soluble protein; it is less straightforward to discern between soluble folded and soluble misfolded protein species in complex cellular environments. To gain insight into this tangled dynamic, Liu et al. (Proc. Natl. Acad. Sci., 2014, 111, 4449−4454) present the design, synthesis, and utility of fluorescent small molecule probes that are highly selective for a folded and functional protein-of-interest over their soluble, but misfolded, counterparts. The authors test their approach with two proteins, a de novodesigned retroaldolase and the thyroid hormone binding protein transthyretin. They use mutated, destabilized versions of these proteins, because an increased proportion of the protein-of-interest is present in a soluble misfolded state− facilitating an investigation of the distribution of folded and soluble misfolded proteins in cells. Using small molecule fluorescent folding probes, they were able to quantitate the fraction of folded and functional protein in cell lysates at a given point in time and discover the dependence of this fraction on the cellular folding environment. Key to the success of their approach was the presence of sufficient cellular holdase activity, created by ATP depletion of the lysed cell, which converts chaperones to holdases that bind avidly to protein folding intermediates. Cellular holdase activity prevents folding probeassociated folding equilibrium shifts. This study offers insight into the intricacies that govern cellular proteostasis and 10
DOI: 10.1021/acschembio.5b00012 ACS Chem. Biol. 2015, 10, 7−11
ACS Chemical Biology
Spotlight
non-natural substrates with specificities and efficiencies similar to the wild-type enzyme. Moreover, the new substrates were delivered to the assembly line with high biosynthetic fidelity, leading to the formation of cyclic dipeptides with novel functionality. The authors note that the reprogramming of NRPSs to accept clickable substrates offers a potentially powerful means of modifying biologically active peptides for diverse applications in the future. Heidi A. Dahlmann, Ph.D.
11
DOI: 10.1021/acschembio.5b00012 ACS Chem. Biol. 2015, 10, 7−11
