Spotlight pubs.acs.org/acschemicalbiology
■
■
TOTAL CHROMOSOME SYNTHESIS
MUSCLING UP ON ENGINEERED TISSUE
From Annaluru, N., et al., Science, 2014, 344, 55−58. Reprinted with permission from AAAS.
Probing the consequences of systematically abrogating the function of multiple nonessential genes within specific chromosomes offers insight into intriguing evolutionary and functional questions, such as how many nonessential genes can be eliminated simultaneously. Current genetic manipulation methods do not easily lend themselves to address this specific question. Now, Annaluru et al. (Science 2014, 344, 55−58) report the de novo total synthesis of a yeast chromosome, referred to as synIII, designed to enable the systematic manipulation of nonessential genes. Based on chromosome III of Saccharomyces cerevisiae, synIII was designed in silico and has a number of replacements, deletions, and insertions that distinguish it from its native counterpart. Construction of synIII began with production of 750 base-pair building blocks from overlapping 60- to 79-mer oligonucleotides, which were then assembled into DNA minichunks in vitro, and finally into an entire chromosome by homologous recombination in yeast, replacing the native sequence. Remarkably, the changes in synIII did not significantly affect the fitness, transcriptome, or replication timing of the synIII strain. In addition to being approximately 40,000 base pairs shorter than the native chromosome, SynIII is constructed such that all nonessential genes are flanked by loxPsym sites, which enables inducible evolution and genome reduction, or what the authors call scrambling, in vivo. SynIII was demonstrated to be functional in S. cerevisiae, but scrambling in a heterozygous mating incompetent diploid strain led to a large increase of a-mater derivatives, which likely resulted from the loss of the MATα allele on synIII. This strategy for creating designer chromosomes lays the foundation for the engineering of entire genomes composed of synthetic chromosomes endowed with desired properties and functionality.
Juhas, M., et al., Proc. Natl. Acad. Sci. U.S.A., 111, 5508−5513. Copyright 2014 National Academy of Sciences, U.S.A. Juhas, M., et al., Proc. Natl. Acad. Sci. U.S.A., 111, 5508−5513. Copyright 2014 National Academy of Sciences, U.S.A.
Engineered muscle tissue has numerous potential applications, including its use as a model for understanding muscle physiology and as a treatment for muscular disorders. However, in order to be fully functional, e.g., to engage in long-term survival and to repair injured muscle tissue, engineered muscle must be able to assimilate into the host vascular system as well as increase its functional output after implantation. Now, Juhas et al. (Proc. Natl. Acad. Sci., 2014, 111, 5508−5513) report the development of novel biomimetic engineered muscle with precisely these capabilities. The authors created a platform for engineering muscle tissue and evaluating its function both in vitro and in vivo. Using rat muscle stem cells, they created muscle bundles in vitro that strongly resembled native muscle tissue. In culture, the engineered muscle cells proliferated and differentiated into mature muscle tissue, as assessed by monitoring expression of proliferation markers and myogenic transcription factors. In addition, the engineered muscle was functional, indicated by measuring the contractile forcegenerating capacity of the tissue over time, and was capable of self-repair in response to injury. For in vivo assessment, engineered muscle bundles were implanted in live mice and imaged in real-time. Remarkably, the implanted muscle underwent rapid vascularization and perfusion with the host blood vessels. Moreover, the bundles continued growth and differentiation, and functionality was demonstrated using a calcium sensor and by measuring active force generation. This study presents a significant step forward in the development of engineered muscle tissue for biotechnological and therapeutic applications. Eva J. Gordon, Ph.D.
Eva J. Gordon, Ph.D.
Published: May 16, 2014 © 2014 American Chemical Society
1067
dx.doi.org/10.1021/cb500342r | ACS Chem. Biol. 2014, 9, 1067−1069
ACS Chemical Biology
■
Spotlight
SORTING OUT PROTEIN FUNCTION
This study illuminates the potential of SORT-M to investigate diverse biological processes, including development, differentiation, and morphogenesis. In addition, this versatile strategy can be adapted to utilize different tags and extended to the study of other organisms. Eva J. Gordon, Ph.D.
■
KINASE TURNS UP THE TRANSLATION
Reprinted from Cell, 157, Martin, I., et al., Ribosomal Protein s15 Phosphorylation Mediates LRRK2 Neurodegeneration in Parkinson’s Disease, 472−485. Copyright 2014, with permission from Elsevier. Reprinted from Cell, 157, Martin, I., et al., Ribosomal Protein s15 Phosphorylation Mediates LRRK2 Neurodegeneration in Parkinson’s Disease, 472−485. Copyright 2014, with permission from Elsevier.
Reprinted by permission from Macmillan Publishers Ltd.: Nature Biotechnology, advance online publication, 13 April 2014, DOI: 10.1038/nbt.2860.
A decade ago, familial cases of Parkinson’s disease were linked to a specific kinase activity in the brain. The characteristic neurodegeneration of the disease was linked to mutations in the leucine-rich repeat kinase 2, LRRK2. Several independent mutations in LRRK2 were uncovered in patients and they all displayed enhanced kinase activity. To understand why this enzyme’s increased activity is pathogenic, Martin et al. (Cell 2014, 157, 472−485) used an affinity purification strategy to hunt for proteins that interact with LRRK2. After enriching for phosphopeptides, mass spectrometry identified a variety of proteins, many of which were components of the ribosome. Screening the list of proteins by in vitro assays helped narrow down the list to just those substrate proteins displaying higher phosphorylation when incubated with pathogenic LRRK2 proteins as compared to the wild-type. Among the short list was a component of the 40S ribosomal subunit, s15 and further experiments traced the phosphorylation event to one specific threonine, 136. But could a phosphorylation event on a ribosomal protein contribute to the neurodegeneration seen in the disease? To pursue that question, the researchers used both mammalian neuron cell culture and a Drosophila model of the disease to test whether pathogenic LRRK2 mutants indeed exerted their toxicity through s15 by threonine 136 phosphorylation. Mutation of residue 136 on s15 to the more inert alanine was protective to neurons in the fly and in both human and rat cell culture. In contrast, swapping the same s15 residue to an aspartic acid to mimic the negative charge of a phosphate modification displayed similar toxicity to when pathogenic LRRK2 was expressed. Finally, increased phosphorylation of
Reprinted by permission from Macmillan Publishers Ltd.: Nature Biotechnology, advance online publication, 13 April 2014, DOI: 10.1038/nbt.2860.
Methods to identify specific proteins synthesized at specific times in live animals provide valuable insight into the diverse and dynamic processes that guide cellular function. Though various approaches have been developed with this goal in mind, each has limitations that hinder its application and/or scope. For example, some approaches enable tagging of a only single site on a single protein, and many require that the cells are grown in minimal media. Now, Elliott et al. (Nature Biotechnology, advance online publication April 13, 2014; DOI: 10.1038/nbt.2860) present stochastic orthogonal recoding of translation with chemoselective modification (SORT-M), a method for temporally and site-specifically tagging the proteome. In SORT-M, chemically modifiable amino acid analogues are incorporated into the proteome at diverse codons by an orthogonal pyrrolysyl-tRNA synthetase/tRNA pair expressed in the cell. Specifically, cyclopropene-containing amino acids, which react with tetrazine probes via an inverse electron demand Diels−Alder cycloaddition reaction, were utilized to label proteomes in bacterial, insect, and mammalian cells. The authors demonstrate the utility of SORT-M for multiple applications, including tagging newly synthesized proteins; labeling the proteome at multiple codon sites; labeling the proteome of cells grown in rich media; and labeling and identifying proteins in specific tissues and defined developmental stages in live fruit flies. 1068
dx.doi.org/10.1021/cb500342r | ACS Chem. Biol. 2014, 9, 1067−1069
ACS Chemical Biology
Spotlight
s15 increased both cap-dependent and cap-independent translation so it appears to be causing a general increase in translation. There may still be other important targets for this kinase, but these results demonstrate that a protein that may have been considered a “housekeeping gene” can easily be the target for a pathogenic enzyme to cause a significant phenotype. Jason G. Underwood, Ph.D.
■
CHARGING AGAINST ANTIBIOTIC RESISTANCE
Reprinted with permission from Zhang, J. et al., J. Am. Chem. Soc., 136, 4873−4876. Copyright 2014 American Chemical Society.
As increasing bacterial resistance to antibiotics continues to plague the medical community, the development of new antibiotics has not kept up with the problem. Methicillinresistant Staphylococcus aureus (MRSA), Gram-positive bacterial strains resistant to multiple conventional antibiotics, pose a particular health threat. As one of their chief resistance mechanisms, these microbes release β-lactamase, an enzyme that hydrolyses the core β-lactam moiety at the core of many antibiotics. Now Zhang et al. (J. Am. Chem. Soc. 2014, 136, 4873− 4876) report that new cationic cobalt polymers may provide a new strategy for combating this resistance. Zhang et al. decided to test new synthetic polymers that include cationic cobaltocenium groups that pair well with carboxylate anions. By complexing with the carboxylate anions on the antibiotics, the researchers suspected that they could prevent β-lactamase from deactivating the drugs. They initially dissolved a series of the polymers with different anionic counterions in solution with nitrocefin, a antibiotic that changes color from yellow to red when its β-lactam is hydrolyzed. When these solutions were incubated with a hospital-based MRSA strain, all solutions with water-soluble polymer remained yellow, showing that they effectively inhibited β-lactamase hydrolysis. The researchers saw similar effects with other β-lactam antibiotics. In disk diffusion assays, the antibiotic-polymer combination inhibited the growth of various MRSA cells, but some strains were not as effectively inhibited, suggesting that resistance mechanisms other than β-lactamase may be at work in those organisms. Ion exchange studies also showed that the polymers bind effectively to anionic polymers that mimic the negative charge of the cell wall. In addition, the metallopolymers alone killed 90% of bacteria at 5 μM concentrations. Previously cationic polymers have been relatively toxic to cells, but these cobaltocenium polymers do not lyse red blood cells. The combination of the antimicrobial activity of these polymers and their apparent low toxicity suggests that this platform could be useful for developing new ways to combat resistant bacteria. Sarah A. Webb, Ph.D. 1069
dx.doi.org/10.1021/cb500342r | ACS Chem. Biol. 2014, 9, 1067−1069
