Articles in 2014

Bioretrosynthesis is meant to simplify construction of metabolic pathways by screening only for the final desired product. This approach, aided by protein design and crystallography, is now used to synthesize an antiretroviral nucleoside analog and surprisingly identifies a new enzyme function.

Host cells respond to bacterial infection by producing itaconate, an inhibitor of bacterial metabolism, among other strategies. Biochemical characterization now defines genes known to be important for bacterial virulence as a new pathway that degrades itaconate into metabolic building blocks.

A new small-molecule inhibitor of the Hedgehog signaling pathway acts independently of Smoothened (Smo). Rather than acting through Smo, which is a GPCR-like protein of the pathway, the compound acts through the orphan GPCR GPR39, with the level of GPR39 activation correlating with Hh pathway inhibition.

Biophysical analysis reveals that conserved G-rich sequences within the mRNAs of gammaherpesvirus genome maintenance proteins (GMPs) form G-quadruplexes (G4). Stabilization of mRNA G4 motifs represses GMP translation, whereas destabilization enhances translation, suggesting that these RNA elements are cis-acting translational regulators of proteins involved in viral latency.

XPB and XPD are essential helicases with roles in transcription and DNA repair. Genomewide ChIP analysis revealed that XPB and XPD localize to DNA G-quadruplex sequences, including many at the transcriptional start sites of actively expressed genes, suggesting that these alternative DNA structures may serve as genome regulatory elements.

A coupled activation-dimerization approach to dissect which of the diverse signaling pathways downstream of Src kinase are responsible for its range of functions such as cell spreading and filopodia formation reveals different roles for focal adhesion kinase and p130Cas.

The use of CoA thioester intermediates drives formation of small- and medium-sized esters in metabolically engineered E. coli cells, including doubly branched chains generated with enzymes from amino acid degradation pathways.

Kinases are a widely targeted enzyme class in cancer chemotherapy. Several clinically used kinase inhibitors also inhibit bromodomains, epigenetic ‘readers’ of acetylated lysine residues, suggesting that kinase-bromodomain polypharmacology may offer benefits in therapeutic settings.

Nematodes are known to stand on their tails and wave—a process called nictation—in an effort to find a new host. Studies of Pristionchus pacificus now show these worms can aggregate to nictate collectively, mediated by the newly discovered natural lipid, nematoil.

A tethered ligand approach reveals that four ligand molecules are required for full desensitization of tetrameric iGluR (glutamate receptor) channels. When fewer ligands are bound, which might be the case during synaptic transmission, desensitization is incomplete or non-existent.

Deubiquitinases (DUBs) are peptidases that remove ubiquitin from post-translationally modified proteins. The identification of a selective small-molecule inhibitor of the USP1–UAF1 deubiquitination complex reveals a role for deubiquitination in regulating the DNA damage response.

Synthetic analogs of nocardicin G—a key precursor of β-lactam antibiotics—are used to show that construction of this enigmatic modified tripeptide relies on an unusual thioesterase domain that epimerizes one residue of an intermediate pentapeptide, but only when the lactam ring is already formed.

Oligonucleotide-based RNA targeting is facilitated by base pairing rules, but identifying small molecules that uniquely bind a specific RNA sequence has been challenging. Inforna, an RNA sequence–based lead optimization strategy, was developed and applied to identify small molecules that inhibit pre-microRNA processing.

A nontargeted metabolomics approach finds that ubiquinone (Q8) accumulates in E. coli with sustained hyperosmotic stress. A new role for Q8 in stress tolerance does not involve its known roles in radical scavenging or as a respiratory electron carrier.

Phage-assisted continuous evolution (PACE) minimizes researcher intervention while maximizing rounds of protein evolution. New strategies now eliminate the need for intermediate substrate analogs and promote altered selectivity instead of promiscuity, exemplified by a 10,000-fold switch in polymerase specificity while retaining wild-type activity.

Halogenases differ from hydroxylases by coordination of a chloride ion at the reactive iron center, which is taken up by an activated substrate. Biochemical and spectroscopic evidence now show other anions can be used, resulting in the first enzymatic incorporation of nitrogen onto unactivated aliphatic carbons.

A tethered ligand approach reveals that calcium-binding proteins (CaBPs) act as allosteric modulators of calcium channel calmodulin regulation, shedding light on how trace CaBPs can prevail over an abundance of CaM.

An NMR analysis of RAS-binding domains (effectors) placed under direct competition allows a systems-level view of effector binding and describes how oscillating concentrations can lead to effector switching. Deconvolution of effector binding to oncogenic RAS^G12V revealed a reordering of the effector hierarchy.

Optogenetic systems permit the temporal and spatial control of gene expression using light. A variant of the LOV domain–containing EL222 protein displays responsive blue light–gated transcriptional control of genes in zebrafish and in mammalian cell lines.

Synthetic biology requires orthogonal inputs and outputs to avoid undesired crosstalk between genetic constructs. Transcription activator–like effectors (TALEs), which bind diverse DNA sequences and can thus be orthogonal, are now employed to construct NOR gates and logic circuits in mammalian cells.