Ribosomal incorporation of cyclic ß-amino acids into peptides using in vitro translation.

We demonstrate in vitro incorporation of cyclic ß-amino acids into peptides by the ribosome through genetic code reprogramming. Further, we show that incorporation efficiency can be increased through the addition of elongation factor P.

Ribosomal Elongation of Cyclic γ-Amino Acids using a Reprogrammed Genetic Code.

Because γ-amino acids generally undergo rapid self-cyclization upon esterification on the carboxyl group, for example, γ-aminoacyl-tRNA, there are no reports of the ribosomal elongation of γ-amino acids to the best of our knowledge. To avoid such self-cyclization, we utilized cyclic γ-amino acids and demonstrated their elongation into a peptide chain. Although the incorporation of the cyclic γ-amino acids is intrinsically slow, we here show that the combination of elongation factor P and engineered tRNAs improves cyclic γ-amino acid incorporation efficiency. Via this method, thioether-macrocyclic peptides containing not only cyclic γ-amino acids but also d-α-, N-methyl-α-, and cyclic ß-amino acids were expressed under the reprogrammed genetic code. Ribosomally synthesized macrocyclic peptide libraries containing cyclic γ-amino acids should be applicable to in vitro screening methodologies such as mRNA display for discovering novel peptide drugs.

Expression and Regulation of PpEIN3b during Fruit Ripening and Senescence via Integrating SA, Glucose, and ACC Signaling in Pear (Pyrus pyrifolia Nakai. Whangkeumbae).

The economic value of fruit is reduced by having a short shelf life. Whangkeumbae is a type of sand pear (Pyrus pyrifolia) considered a climacteric fruit. The pear is famous for its smooth surface and good flavor. However, its shelf life is very short because of senescence and disease after harvest and a burst of ethylene (ET) production prompting the onset of fruit ripening. In plants, ETHYLENE INSENSITIVE3 (EIN3) and EIN3like (EIL), located in the nucleus, are important components of the ET signaling pathway and act as transcription factors. EIN3s and EILs belong to a small family involved in regulating the expression of ethylene response factor gene (ERF), whose encoding protein is the final component in the ET signaling pathway. The mutation of these components will cause defects in the ethylene pathway. In this study, one gene encoding an EIN3 was cloned and identified from Whangkeumbae and designated PpEIN3b. The deduced PpEIN3b contained a conserved EIN3 domain, a bipartite nuclear localization signal profile (NLS_BP), and an N-6 adenine-specific DNA methylase signature (N6_MTASE). PpEIN3b belongs to the EIN3 super-family by phylogenetic analysis. Quantitative RT-PCR (qRT-PCR) analysis revealed that PpEIN3b was preferentially expressed in fruit. Additionally, its expression was developmentally regulated during fruit ripening and senescence. Furthermore, PpEIN3b transcripts were obviously repressed by salicylic acid (SA) and glucose treatment in pear fruit and in diseased fruit, while it was significantly induced by 1-aminocyclopropane-1-carboxylic acid (ACC) treatment. Taken together, our results reveal the expression and regulation profiles of PpEIN3b and suggest that PpEIN3b might integrate SA, glucose, and ACC signaling to regulate fruit ripening and senescence in pear, which would provide a candidate gene for this regulation to obtain fruit with a long shelf life and improved economic value.

Disruption and Overexpression of the Gene Encoding ACC (1-Aminocyclopropane-1-Carboxylic Acid) Deaminase in Soil-Borne Fungal Pathogen Verticillium dahliae Revealed the Role of ACC as a Potential Regulator of Virulence and Plant Defense.

It has been suggested that some microorganisms, including plant growth-promoting rhizobacteria, manipulate the level of ethylene in plants by degrading 1-aminocyclopropane-1-carboxylic acid (ACC), an ethylene precursor, into α-ketobutyrate and ammonia, using ACC deaminase (ACCd). Here, we investigated whether ACCd of Verticillium dahliae, a soil-borne fungal pathogen of many important crops, is involved in causing vascular wilt disease. Overexpression of the V. dahliae gene encoding this enzyme, labeled as ACCd, significantly increased virulence in both tomato and eggplant, while disruption of ACCd reduced virulence. Both types of mutant produced more ethylene than a wild-type (70V-WT) strain, although they significantly differed in ACC content. Overexpression strains lowered ACC levels in the roots of infected plants, while the amount of ACC in the roots of plants infected with deletion mutants increased. To test the hypothesis that ACC acts as a signal for controlling defense, roots of WT and Never-ripe (Nr) tomato plants were treated with ACC before V. dahliae inoculation. Plants pretreated with ACC displayed less severe symptoms than untreated controls. Collectively, our results suggest a novel role of ACC as a regulator of both plant defense and pathogen virulence.

Expression of an exogenous 1-aminocyclopropane-1-carboxylate deaminase gene in psychrotolerant bacteria modulates ethylene metabolism and cold induced genes in tomato under chilling stress.

The role of stress induced ethylene under low temperature stress has been controversial and hitherto remains unclear. In the present study, 1-aminocyclopropane-1-carboxylate deaminase (ACCD) gene, acdS expressing mutant strains were generated from ACCD negative psychrotolerant bacterial strains Flavobacterium sp. OR306 and Pseudomonas frederiksbergensis OS211, isolated from agricultural soil during late winter. After transformation with plasmid pRKACC which contained the acdS gene, both the strains were able to exhibit ACCD activity in vitro. The effect of this ACCD under chilling stress with regards to ethylene was studied in tomato plants inoculated with both acdS expressing and wild type bacteria. On exposing the plants to one week of chilling treatment at 12/10 °C, it was found that stress ethylene, ACC accumulation and ACO activity which are markers of ethylene stress, were significantly reduced in plants inoculated with the acdS gene transformed mutants. In case of plants inoculated with strain OS211-acdS, ethylene emission, ACC accumulation and ACO activity was significantly reduced by 52%, 75.9% and 23.2% respectively compared to uninoculated control plants. Moreover, expression of cold induced LeCBF1 and LeCBF3 genes showed that these genes were significantly induced by the acdS transformed mutants in addition to reduced expression of ethylene-responsive transcription factor 13 (ETF-13) and ACO genes. Induced expression of LeCBF1 and LeCBF3 in plants inoculated with acdS expressing mutants compared to wild type strains show that physiologically evolved stress ethylene and its transcription factors play a role in regulation of cold induced genes as reported earlier in the literature.

TR-DB: an open-access database of compounds affecting the ethylene-induced triple response in Arabidopsis.

Small molecules which act as hormone agonists or antagonists represent useful tools in fundamental research and are widely applied in agriculture to control hormone effects. High-throughput screening of large chemical compound libraries has yielded new findings in plant biology, with possible future applications in agriculture and horticulture. To further understand ethylene biosynthesis/signaling and its crosstalk with other hormones, we screened a 12,000 compound chemical library based on an ethylene-related bioassay of dark-grown Arabidopsis thaliana (L.) Heynh. seedlings. From the initial screening, 1313 (∼11%) biologically active small molecules altering the phenotype triggered by the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC), were identified. Selection and sorting in classes were based on the angle of curvature of the apical hook, the length and width of the hypocotyl and the root. A MySQL-database was constructed (https://chaos.ugent.be/WE15/) including basic chemical information on the compounds, images illustrating the phenotypes, phenotype descriptions and classification. The research perspectives for different classes of hit compounds will be evaluated, and some general screening tips for customized high-throughput screening and pitfalls will be discussed.

Physiological response to drought in radiata pine: phytohormone implication at leaf level.

Pinus radiata D. Don is one of the most abundant species in the north of Spain. Knowledge of drought response mechanisms is essential to guarantee plantation survival under reduced water supply as predicted in the future. Tolerance mechanisms are being studied in breeding programs, because information on such mechanisms can be used for genotype selection. In this paper, we analyze the changes of leaf water potential, hydraulic conductance (K(leaf)), stomatal conductance and phytohormones under drought in P. radiata breeds (O1, O2, O3, O4, O5 and O6) from different climatology areas, hypothesizing that they could show variable drought tolerance. As a primary signal, drought decreased cytokinin (zeatin and zeatin riboside-Z + ZR) levels in needles parallel to K(leaf) and gas exchange. When Z + ZR decreased by 65%, indole-3-acetic acid (IAA) and abscisic acid (ABA) accumulation started as a second signal and increments were higher for IAA than for ABA. When plants decreased by 80%, Z + ZR and K(leaf) doubled their ABA and IAA levels, the photosystem II yield decreased and the electrolyte leakage increased. At the end of the drought period, less tolerant breeds increased IAA over 10-fold compared with controls. External damage also induced jasmonic acid accumulation in all breeds except in O5 (P. radiata var. radiata × var. cedrosensis), which accumulated salicylic acid as a defense mechanism. After rewatering, only the most tolerant plants recovered their K(leaf,) perhaps due to an IAA decrease and 1-aminocyclopropane-1-carboxylic acid maintenance. From all phytohormones, IAA was the most representative 'water deficit signal' in P. radiata.

Transcriptome profiling, molecular biological, and physiological studies reveal a major role for ethylene in cotton fiber cell elongation.

Upland cotton (Gossypium hirsutum) produces the most widely used natural fibers, yet the regulatory mechanisms governing fiber cell elongation are not well understood. Through sequencing of a cotton fiber cDNA library and subsequent microarray analysis, we found that ethylene biosynthesis is one of the most significantly upregulated biochemical pathways during fiber elongation. The 1-Aminocyclopropane-1-Carboxylic Acid Oxidase1-3 (ACO1-3) genes responsible for ethylene production were expressed at significantly higher levels during this growth stage. The amount of ethylene released from cultured ovules correlated with ACO expression and the rate of fiber growth. Exogenously applied ethylene promoted robust fiber cell expansion, whereas its biosynthetic inhibitor l-(2-aminoethoxyvinyl)-glycine (AVG) specifically suppressed fiber growth. The brassinosteroid (BR) biosynthetic pathway was modestly upregulated during this growth stage, and treatment with BR or its biosynthetic inhibitor brassinazole (BRZ) also promoted or inhibited, respectively, fiber growth. However, the effect of ethylene treatment was much stronger than that of BR, and the inhibitory effect of BRZ on fiber cells could be overcome by ethylene, but the AVG effect was much less reversed by BR. These results indicate that ethylene plays a major role in promoting cotton fiber elongation. Furthermore, ethylene may promote cell elongation by increasing the expression of sucrose synthase, tubulin, and expansin genes.

Molecular determinants of ligand binding to the human melanocortin-4 receptor.

To elucidate the molecular basis for the interaction of ligands with the human melanocortin-4 receptor (hMC4R), agonist structure-activity studies and receptor point mutagenesis were performed. Structure-activity studies of [Nle(4), D-Phe(7)]-alpha-melanocyte stimulating hormone (NDP-MSH) identified D-Phe7-Arg8-Trp9 as the minimal NDP-MSH fragment that possesses full agonist efficacy at the hMC4R. In an effort to identify receptor residues that might interact with amino acids in this tripeptide sequence 24 hMC4R transmembrane (TM) residues were mutated (the rationale for choosing specific receptor residues for mutation is outlined in the Results section). Mutation of TM3 residues D122 and D126 and TM6 residues F261 and H264 decreased the binding affinity of NDP-MSH 5-fold or greater, thereby identifying these receptor residues as sites potentially involved in the sought after ligand-receptor interactions. By examination of the binding affinities and potencies of substituted NDP-MSH peptides at receptor mutants, evidence was found that core melanocortin peptide residue Arg8 interacts at a molecular level with hMC4R TM3 residue D122. TM3 mutations were also observed to decrease the binding of hMC4R antagonists. Notably, mutation of TM3 residue D126 to alanine decreased the binding affinity of AGRP (87-132), a C-terminal derivative of the endogenous melanocortin antagonist, 8-fold, and simultaneous mutations D122A/D126A completely abolished AGRP (87-132) binding. In addition, mutation of TM3 residue D122 or D126 decreased the binding affinity of hMC4R antagonist SHU 9119. These results provide further insight into the molecular determinants of hMC4R ligand binding.

Aromatic amino acids in region 2.3 of Escherichia coli sigma 70 participate collectively in the formation of an RNA polymerase-promoter open complex.

Formation of an initiation-competent RNA polymerase-promoter complex involves DNA melting over a region of about 12 base-pairs, which includes the start site of transcription, thus enabling the template strand to base-pair with the initiating nucleoside triphosphates. By studying the effects of alanine substitutions, we have investigated the role of the aromatic amino residues in the Escherichia coli sigma(70) conserved region 2.3 in promoter strand separation. The resulting mutants were assessed for their activity in vivo in the context of a sigma(70)/sigma(32) hybrid sigma factor that could be targeted to a specific hybrid promoter in the cell. All substitutions lead to an at least twofold reduction in expression of the hybrid promoter-driven reporter gene. The in vitro assay of single substitutions indicated cold sensitivity similar to that previously observed with analogous substitutions in Bacillus subtilis sigma(A). Kinetic assays showed that these substitutions slowed the rate of open complex formation at 37 degrees C as well. RNA polymerase reconstituted with a sigma(70) containing multiple alanine substitutions readily binds to promoter DNA, but then proceeds slowly beyond the first intermediate complex on the pathway to formation of the transcription-competent complex. These data demonstrate that together the aromatic residues in region 2.3 of E. coli sigma(70) ensure that DNA strand separation proceeds efficiently, even if no individual residue may be essential for accomplishment of the process.

How the substitution of K188 of trypsin binding site by aromatic amino acids can influence the processing of beta-casein.

Aspartyl 189 residue of trypsin is known to be essential for specific lysis of Arg-X and Lys-X bonds. Undertaking to modulate the catalytic properties of this protease, otherwise highly conserved K188 was replaced with aromatic amino acid residues aiming the perturbation of the electrostatics and the amplifying of hydrophobic interactions of the substrate binding site. The catalytic properties of the mutants K188F, K188Y, and K188W were measured at pH 7, 8, 9, and 10 using a pair of synthetic tetrapeptide p-nitroanilide substrates and beta-casein. The kinetic analysis reveals that all the mutants conserve the native trypsin capacity to split peptide bonds containing arginyl and lysyl residues. Surprisingly, however, depending on mutation, the optimum pH of activity changes. As demonstrated only by proteolysis of a natural substrate, all mutants cleave also peptide bonds involving asparagine and glutamine. These stuttered cleavage sites are close to the beta-casein fragments in beta-sheet according to Hydrophobic Cluster Analysis.

Substitutions of conserved aromatic amino acid residues in subunit I perturb the metal centers of the Escherichia coli bo-type ubiquinol oxidase.

Cytochrome bo is a four-subunit quinol oxidase in the aerobic respiratory chain of Escherichia coli and functions as a redox-coupled proton pump. Subunit I binds all the redox metal centers, low-spin heme b, high-spin heme o, and CuB, whose axial ligands have been identified to be six invariant histidines. This work explored the possible roles of the aromatic amino acid residues conserved in the putative transmembrane helices (or at the boundary of the membrane) of subunit I. Sixteen aromatic amino acid residues were individually substituted by Leu, except for Tyr61 and Trp282 by Phe and Phe415 by Trp. Leu substitutions of Trp280 and Tyr288 in helix VI, Trp331 in loop VII-VIII, and Phe348 in helix VIII reduced the catalytic activity, whereas all other mutations did not affect the in vivo activity. Spectroscopic analyses of the purified mutant enzymes revealed that the defects were attributable to perturbations of the binuclear center. On the basis of these findings and recent crystallographic studies on cytochrome c oxidases, we discuss the possible roles of the conserved aromatic amino acid residues in subunit I of the heme-copper terminal oxidases.

A cluster of aromatic residues in the sixth membrane-spanning segment of the dopamine D2 receptor is accessible in the binding-site crevice.

The binding site of the dopamine D2 receptor, like that of other homologous G protein-coupled receptors, is contained within a water-accessible crevice formed among its seven membrane-spanning segments. Using the substituted-cysteine accessibility method, we previously mapped the residues in the third, fifth, and seventh membrane-spanning segments that contribute to the surface of this binding-site crevice. We have now mutated to cysteine, one at a time, 22 consecutive residues in the sixth membrane-spanning segment (M6) and expressed the mutant receptors in HEK 293 cells. Ten of these mutants reacted with charged, hydrophilic, lipophobic, sulfhydryl-specific reagents, added extracellularly, and all but one were protected from reaction by a reversible dopamine antagonist, sulpiride. Thus, we infer that the side chains of the residues at the reactive loci (V378, F382, W386, P388, F389, F390, T392, H393, I394, and I397) are on the water-accessible surface of the binding-site crevice. The pattern of accessibility is consistent with an alpha-helical conformation with a wide angle of accessibility near the binding site itself and a narrower stripe continuing toward the cytoplasmic portion of the binding-site crevice. This pattern of accessibility is consistent with the presence of a proline kink which could bend the extracellular portion of M6 into the binding-site crevice where it would be more broadly accessible than the cytoplasmic portion of the membrane-spanning segment. Four highly conserved aromatic residues and a histidine are clustered together on the water-accessible surface of the binding-site crevice. They define an interconnected "aromatic cluster" that may be involved in ligand binding and receptor activation.