No abstract

The study of epigenetics has greatly benefited from the development and application of various chemical biology approaches. In this review, we highlight the key targets for modulation and recent methods developed to enact such modulation. We discuss various chemical biology techniques to study DNA methylation and the post-translational modification of histones as well as their effect on gene expression. Additionally , we address the wealth of protein synthesis approaches to yield histones and nucleosomes bearing epigenetic modifications. Throughout, we highlight targets that present opportunities for the chemical biology community, as well as exciting new approaches that will provide additional insight into the roles of epigenetic marks.

The transport of leucine in the apical-to-basal (retina to choroid) direction across the isolated bovine retinal pigment epithelium is mediated predominantly by the L amino transport system at low carrier (10 microns) concentrations. There is no evidence of an active or facilitated transport system operating in the opposite direction. The identification of the L system is based on the lack of sodium dependence, specific inhibition of leucine transport by 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH), and the demonstration of trans-stimulation. Lysine, glutamate, and 2-methylaminoisobutyric acid (MeAIB) did not provide any competitive inhibition. Ouabain and iodoacetate were also ineffective in modifying leucine transport. The transport mediated by the L system was markedly temperature sensitive, whereas no temperature dependence was apparent in the transport of leucine in the basal-to-apical direction (choroid to retina). When treated with dinitrophenol (DNP), the transport of leucine in the apical-to-basal direction was greatly enhanced, but no effect was observed on the leucine movement in the opposite direction. Azide and rotenone had an effect similar to DNP, as did reducing the partial pressure of O2 to less than 40 Torr. The enhancement of transport appeared to be mediated by the activation of an ancillary system, since it was susceptible to different classes of metabolic and competitive inhibitors as well as the observed ionic dependency. After DNP treatment, the transport of leucine was inhibited by lysine and BCH, revealed a sodium dependence, and could be inhibited by iodoacetate. The characteristics of the enhanced transport appear to be similar to those of the recently described G system(s) of amino acid transport.

The site‐specific incorporation of non‐standard and novel amino acid structures enabled by Genetic Code Expansion (GCE) provides access to new avenues of protein research allowed by engineering diverse functionality into proteins. At its core, GCE requires the engineering of an orthogonal amino acyl‐tRNA synthetase/tRNA cognate pair that can function with high catalytic efficiency for a non‐canonical amino acid (ncAA). While the GCE community has generated many new amino acyl‐tRNA synthetase/tRNA pairs, these pairs have kinetics that are orders of magnitude worse than the natural translational components. A major challenge for the GCE field is identifying which amino acyl‐tRNA synthetase residues to target for engineering improved amino acyl‐tRNA synthetase/tRNA pairs.Here we use Rosetta modeling to identify mutations in the amino acyl‐tRNA synthetase active site that will improve the efficiency of the nitroTyr amino acyl‐tRNA synthetase/tRNA pairs. Rosetta guided mutations were successful in generating improved GCE function in vivo.Crystal structures were obtained for the Rosetta improved amino acyl‐tRNA synthetases to understand the structural basis for the enhanced activity as part of a comprehensive kinetic and structural analysis of these improved synthetases. Here, we used the 3‐nitro‐tyrosine amino acyl‐tRNA synthetase/tRNA pair as a model system for synthetase improvement and structural characterization.We observe that the Rosetta‐improved synthetases allow a more planar 3‐nitro‐tyrosine (nitroTyr) amino acid upon binding, which is predicted to contribute to their increased activity. This Rosetta guided approach provides a third generation of more efficient nitroTyr amino acyl‐tRNA synthetase/tRNA pairs that will accelerate the study of this oxidative stress post‐translational modification and its role in human disease.We demonstrate that Rosetta successfully identified key residues for amino acyl‐tRNA synthetase/tRNA pair optimization. This strategy can be utilized to systematically improve the efficiency of all ncAA amino acyl‐tRNA synthetase/tRNA pairs, resulting in more robust GCE technologies and facilitating new applications.Support or Funding InformationNIH Grant #R01‐GM114653‐01, Oregon State University Summer Undergraduate Research Experience (SURE) in Science ProgramThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.