Editing of misaminoacylated tRNA controls the sensitivity of amino acid stress responses in Saccharomyces cerevisiae.

Amino acid starvation activates the protein kinase Gcn2p, leading to changes in gene expression and translation. Gcn2p is activated by deacylated tRNA, which accumulates when tRNA aminoacylation is limited by lack of substrates or inhibition of synthesis. Pairing of amino acids and deacylated tRNAs is catalyzed by aminoacyl-tRNA synthetases, which use quality control pathways to maintain substrate specificity. Phenylalanyl-tRNA synthetase (PheRS) maintains specificity via an editing pathway that targets non-cognate Tyr-tRNAPhe. While the primary role of aaRS editing is to prevent misaminoacylation, we demonstrate editing of misaminoacylated tRNA is also required for detection of amino acid starvation by Gcn2p. Ablation of PheRS editing caused accumulation of Tyr-tRNAPhe (5%), but not deacylated tRNAPhe during amino acid starvation, limiting Gcn2p kinase activity and suppressing Gcn4p-dependent gene expression. While the PheRS-editing ablated strain grew 50% slower and displayed a 27-fold increase in the rate of mistranslation of Phe codons as Tyr compared to wild type, the increase in mistranslation was insufficient to activate an unfolded protein stress response. These findings show that during amino acid starvation a primary role of aaRS quality control is to help the cell mount an effective stress response, independent of the role of editing in maintaining translational accuracy.

MS-READ: Quantitative measurement of amino acid incorporation.

Ribosomal protein synthesis results in the genetically programmed incorporation of amino acids into a growing polypeptide chain. Faithful amino acid incorporation that accurately reflects the genetic code is critical to the structure and function of proteins as well as overall proteome integrity. Errors in protein synthesis are generally detrimental to cellular processes yet emerging evidence suggest that proteome diversity generated through mistranslation may be beneficial under certain conditions. Cumulative translational error rates have been determined at the organismal level, however codon specific error rates and the spectrum of misincorporation errors from system to system remain largely unexplored. In particular, until recently technical challenges have limited the ability to detect and quantify comparatively rare amino acid misincorporation events, which occur orders of magnitude less frequently than canonical amino acid incorporation events. We now describe a technique for the quantitative analysis of amino acid incorporation that provides the sensitivity necessary to detect mistranslation events during translation of a single codon at frequencies as low as 1 in 10,000 for all 20 proteinogenic amino acids, as well as non-proteinogenic and modified amino acids. This article is part of a Special Issue entitled "Biochemistry of Synthetic Biology - Recent Developments" Guest Editor: Dr. Ilka Heinemann and Dr. Patrick O'Donoghue.

Evolution of translation machinery in recoded bacteria enables multi-site incorporation of nonstandard amino acids.

Expansion of the genetic code with nonstandard amino acids (nsAAs) has enabled biosynthesis of proteins with diverse new chemistries. However, this technology has been largely restricted to proteins containing a single or few nsAA instances. Here we describe an in vivo evolution approach in a genomically recoded Escherichia coli strain for the selection of orthogonal translation systems capable of multi-site nsAA incorporation. We evolved chromosomal aminoacyl-tRNA synthetases (aaRSs) with up to 25-fold increased protein production for p-acetyl-L-phenylalanine and p-azido-L-phenylalanine (pAzF). We also evolved aaRSs with tunable specificities for 14 nsAAs, including an enzyme that efficiently charges pAzF while excluding 237 other nsAAs. These variants enabled production of elastin-like-polypeptides with 30 nsAA residues at high yields (∼50 mg/L) and high accuracy of incorporation (>95%). This approach to aaRS evolution should accelerate and expand our ability to produce functionalized proteins and sequence-defined polymers with diverse chemistries.

Chemical Evolution of a Bacterial Proteome.

We have changed the amino acid set of the genetic code of Escherichia coli by evolving cultures capable of growing on the synthetic noncanonical amino acid L-ß-(thieno[3,2-b]pyrrolyl)alanine ([3,2]Tpa) as a sole surrogate for the canonical amino acid L-tryptophan (Trp). A long-term cultivation experiment in defined synthetic media resulted in the evolution of cells capable of surviving Trp→[3,2]Tpa substitutions in their proteomes in response to the 20,899 TGG codons of the E. coli W3110 genome. These evolved bacteria with new-to-nature amino acid composition showed robust growth in the complete absence of Trp. Our experimental results illustrate an approach for the evolution of synthetic cells with alternative biochemical building blocks.

Genomically recoded organisms expand biological functions.

We describe the construction and characterization of a genomically recoded organism (GRO). We replaced all known UAG stop codons in Escherichia coli MG1655 with synonymous UAA codons, which permitted the deletion of release factor 1 and reassignment of UAG translation function. This GRO exhibited improved properties for incorporation of nonstandard amino acids that expand the chemical diversity of proteins in vivo. The GRO also exhibited increased resistance to T7 bacteriophage, demonstrating that new genetic codes could enable increased viral resistance.

Transfer RNA misidentification scrambles sense codon recoding.

Sense codon recoding is the basis for genetic code expansion with more than two different noncanonical amino acids. It requires an unused (or rarely used) codon, and an orthogonal tRNA synthetase:tRNA pair with the complementary anticodon. The Mycoplasma capricolum genome contains just six CGG arginine codons, without a dedicated tRNA(Arg). We wanted to reassign this codon to pyrrolysine by providing M. capricolum with pyrrolysyl-tRNA synthetase, a synthetic tRNA with a CCG anticodon (tRNA(Pyl)(CCG)), and the genes for pyrrolysine biosynthesis. Here we show that tRNA(Pyl)(CCG) is efficiently recognized by the endogenous arginyl-tRNA synthetase, presumably at the anticodon. Mass spectrometry revealed that in the presence of tRNA(Pyl)(CCG), CGG codons are translated as arginine. This result is not unexpected as most tRNA synthetases use the anticodon as a recognition element. The data suggest that tRNA misidentification by endogenous aminoacyl-tRNA synthetases needs to be overcome for sense codon recoding.

ScanRanker: Quality assessment of tandem mass spectra via sequence tagging.

In shotgun proteomics, protein identification by tandem mass spectrometry relies on bioinformatics tools. Despite recent improvements in identification algorithms, a significant number of high quality spectra remain unidentified for various reasons. Here we present ScanRanker, an open-source tool that evaluates the quality of tandem mass spectra via sequence tagging with reliable performance in data from different instruments. The superior performance of ScanRanker enables it not only to find unassigned high quality spectra that evade identification through database search but also to select spectra for de novo sequencing and cross-linking analysis. In addition, we demonstrate that the distribution of ScanRanker scores predicts the richness of identifiable spectra among multiple LC-MS/MS runs in an experiment, and ScanRanker scores assist the process of peptide assignment validation to increase confident spectrum identifications. The source code and executable versions of ScanRanker are available from http://fenchurch.mc.vanderbilt.edu.

High-throughput profiling of formalin-fixed paraffin-embedded tissue using parallel electrophoresis and matrix-assisted laser desorption ionization mass spectrometry.

Analysis of formalin-fixed paraffin-embedded tissues (FFPE) is increasingly recognized as a strategy for the discovery and validation of clinically useful biomarker candidates. Large tissue collections including tissue microarrays (TMAs) are available, but current analytical strategies for their characterization have limited throughput. In this report, we describe a workflow for rapid analysis of hundreds of FFPE tissue specimens. The strategy combines parallel sample processing and on-chip electrophoresis with automated matrix-assisted laser desorption ionzation mass spectrometry (MALDI MS) analysis. The method is optimized for small quantities of clinically valuable tissues allowing detection of hundreds of peptides from a single core in a TMA section. We describe results from the optimization of the method and apply it for the analysis of tissue microarrays containing formalin fixed tissue specimens from human kidney.

Biomarker discovery by imaging mass spectrometry: transthyretin is a biomarker for gentamicin-induced nephrotoxicity in rat.

Adverse drug effects are often associated with pathological changes in tissue. An accurate depiction of the undesired affected area, possibly supported by mechanistic data, is important to classify the effects with regard to relevance for human patients. MALDI imaging MS represents a new analytical tool to directly provide the spatial distribution and the relative abundance of proteins in tissue. Here we evaluate this technique to investigate potential toxicity biomarkers in kidneys of rats that were administered gentamicin, a well known nephrotoxicant. Differential analysis of the mass spectrum profiles revealed a spectral feature at 12,959 Da that strongly correlates with histopathology alterations of the kidney. We unambiguously identified this spectral feature as transthyretin (Ser(28)-Gln(146)) using an innovative combination of tissue microextraction and fractionation by reverse-phase liquid chromatography followed by a top-down tandem mass spectrometric approach. Our findings clearly demonstrate the emerging role of imaging MS in the discovery of toxicity biomarkers and in obtaining mechanistic insights concerning toxicity mechanisms.

Automated acoustic matrix deposition for MALDI sample preparation.

Novel high-throughput sample preparation strategies for MALDI imaging mass spectrometry (IMS) and profiling are presented. An acoustic reagent multispotter was developed to provide improved reproducibility for depositing matrix onto a sample surface, for example, such as a tissue section. The unique design of the acoustic droplet ejector and its optimization for depositing matrix solution are discussed. Since it does not contain a capillary or nozzle for fluid ejection, issues with clogging of these orifices are avoided. Automated matrix deposition provides better control of conditions affecting protein extraction and matrix crystallization with the ability to deposit matrix accurately onto small surface features. For tissue sections, matrix spots of 180-200 microm in diameter were obtained and a procedure is described for generating coordinate files readable by a mass spectrometer to permit automated profile acquisition. Mass spectral quality and reproducibility was found to be better than that obtained with manual pipet spotting. The instrument can also deposit matrix spots in a dense array pattern so that, after analysis in a mass spectrometer, two-dimensional ion images may be constructed. Example ion images from a mouse brain are presented.

Molecular profiling of experimental Parkinson's disease: direct analysis of peptides and proteins on brain tissue sections by MALDI mass spectrometry.

Direct molecular profiling of biological samples using matrix-assisted laser desorption ionization mass spectrometry is a powerful tool for identifying phenotypic markers. In this report, protein profiling was used for the first time to generate peptide and protein profiles of brain tissue sections obtained from experimental Parkinson's disease (unilaterally 6-hydroxydopamine treated rats). The mass spectrometer was used to map the peptide and protein expression directly on 12 microm tissue sections in mass-to-charge (m/z) values, providing the capability of mapping specific molecules of the original sample, that is, localization, intensity and m/z ratio. Several protein expression profile differences were found in the dopamine depleted side of the brain when compared to the corresponding intact side, for example, calmodulin, cytochrome c, and cytochrome c oxidase. An increased ratio of post-translational modifications such as acetylations were found in the striatum of proteins in the dopamine depleted side of the brain. These modifications were decreased after subchronic administration of L-Dopa. The present study shows that unique protein profiles can be obtained in specific brain regions (and subregions) directly on brain tissue sections and allows for the study of complex biochemical processes such as those occurring in experimental Parkinson's disease.

Comparative analysis of estrogenic activity in sewage treatment plant effluents involving three in vitro assays and chemical analysis of steroids.

In this study, we assessed and compared the suitability of three in vitro screening tools for the measurement of estrogenic activity in sewage treatment plant effluents (STPEs). These assays were the yeast estrogen screen (YES), production of zona radiata proteins (ZRPs) in trout hepatocytes, and the induction of reporter gene expression in the transfected rainbow trout gonad cell line RTG-2. Data obtained with the YES were additionally compared with calculated estrogenicity, based on steroid analysis data of the effluents. For comparison purposes, the response of the in vitro systems toward the estrogenic chemicals beta-estradiol, ethinyl estradiol, bisphenol-A, nonylphenol, and octylphenol was assessed. All three assays showed sensitivities in the same order of magnitude in response to the steroid compounds tested, with ZRP production being the least sensitive. Regarding the estrogenic environmental chemicals tested, the RTG-2 assay was more than an order of magnitude more sensitive than the other two assays. Despite their different sensitivities toward selected test chemicals, the three in vitro systems indicated estrogenic activity in the same concentration range for the tested STPEs. Calculated estrogenicity (chemical analysis) and measured estrogenicity (YES) were of the same order of magnitude for the STPEs tested. The present study indicates that all three in vitro systems, with the yeast-based system being the easiest and most robust, are applicable for the screening of estrogenic activity in effluent samples.

Combined biological and chemical assessment of estrogenic activities in wastewater treatment plant effluents.

Five wastewater treatment plant effluents were analyzed for known endocrine disrupters and estrogenicity. Estrogenicity was determined by using the yeast estrogen screen (YES) and by measuring the blood plasma vitellogenin (VTG) concentrations in exposed male rainbow trout (Oncorhynchus mykiss). While all wastewater treatment plant effluents contained measurable concentrations of estrogens and gave a positive response with the YES, only at two sites did the male fish have significantly increased VTG blood plasma concentrations after the exposure, compared to pre-exposure concentrations. Estrone (E1) concentrations ranged up to 51 ng L(-1), estradiol (E2) up to 6 ng L(-1), and ethinylestradiol (EE2) up to 2 ng L(-1) in the 90 samples analyzed. Alkylphenols, alkylphenolmonoethoxylates and alkylphenoldiethoxylates, even though found at microg L(-1) concentrations in effluents from wastewater treatment plants with a significant industrial content, did not contribute much to the overall estrogenicity of the samples taken due to their low relative potency. Expected estrogenicities were calculated from the chemical data for each sample by using the principle of concentration additivity and relative potencies of the various chemicals as determined with the yeast estrogen screen. Measured and calculated estradiol equivalents gave the same order of magnitude and correlated rather well (R(2)=0.6).