Treatment of ARS deficiencies with specific amino acids.

PURPOSE: Recessive cytosolic aminoacyl-tRNA synthetase (ARS) deficiencies are severe multiorgan diseases, with limited treatment options. By loading transfer RNAs (tRNAs) with their cognate amino acids, ARS are essential for protein translation. However, it remains unknown why ARS deficiencies lead to specific symptoms, especially early life and during infections. We set out to increase pathophysiological insight and improve therapeutic possibilities. METHODS: In fibroblasts from patients with isoleucyl-RS (IARS), leucyl-RS (LARS), phenylalanyl-RS-beta-subunit (FARSB), and seryl-RS (SARS) deficiencies, we investigated aminoacylation activity, thermostability, and sensitivity to ARS-specific amino acid concentrations, and developed personalized treatments. RESULTS: Aminoacylation activity was reduced in all patients, and further diminished at 38.5/40 °C (PLARS and PFARSB), consistent with infectious deteriorations. With lower cognate amino acid concentrations, patient fibroblast growth was severely affected. To prevent local and/or temporal deficiencies, we treated patients with corresponding amino acids (follow-up: 1/2-2 2/3rd years), and intensified treatment during infections. All patients showed beneficial treatment effects, most strikingly in growth (without tube feeding), head circumference, development, coping with infections, and oxygen dependency. CONCLUSION: For these four ARS deficiencies, we observed a common disease mechanism of episodic insufficient aminoacylation to meet translational demands and illustrate the power of amino acid supplementation for the expanding ARS patient group. Moreover, we provide a strategy for personalized preclinical functional evaluation.

Production of aminoacyl prolines using the adenylation domain of nonribosomal peptide synthetase with class III polyphosphate kinase 2-mediated ATP regeneration.

An ATP regeneration system is advantageous for industrial processes that are coupled with ATP-dependent enzymes. For ATP regeneration from AMP, a few methods have been reported; however, these methods employ multiple enzymes. To establish an ATP regeneration system using a single enzyme, we focused on class III polyphosphate kinase 2 (class III PPK2) that can synthesize ATP from AMP and polyphosphate. We constructed an ATP regeneration system from AMP using Deipr_1912, a class III PPK2 from Deinococcus proteolyticus NBRC 101906T, coupled with aminoacyl proline (Xaa-Pro) synthesis catalyzed by the adenylation domain of tyrocidine synthetase A (TycA-A). Using this system, 0.87 mM of l-Trp-l-Pro was successfully synthesized from AMP after 72 h. Farther, addition of inorganic pyrophosphatase from Escherichia coli to the coupling reaction increased the reaction rate by 14-fold to yield 6.2 mM l-Trp-l-Pro. When the coupling reaction was applied to whole-cell reactions in E. coli BL21(DE3) pepQ-putA-, ATP was successfully regenerated from AMP by Deipr_1912, and 6.7 mM of l-Trp-l-Pro was produced after 24 h with the supplementation of 10 mM AMP. In addition, by altering the substrate amino acid of TycA-A, not only l-Trp-l-Pro, but also various other l-Xaa-l-Pro (Xaa = Val, Leu, Met, or Tyr) were produced using the whole-cell reaction incorporating ATP regeneration. Therefore, a production method for Xaa-Pro employing the adenylation domain of a nonribosomal peptide synthetase was established by introducing an ATP regeneration system that utilizes class III PPK2 with pyrophosphatase.

Structures of two bacterial resistance factors mediating tRNA-dependent aminoacylation of phosphatidylglycerol with lysine or alanine.

The cytoplasmic membrane is probably the most important physical barrier between microbes and the surrounding habitat. Aminoacylation of the polar head group of the phospholipid phosphatidylglycerol (PG) catalyzed by Ala-tRNA(Ala)-dependent alanyl-phosphatidylglycerol synthase (A-PGS) or by Lys-tRNA(Lys)-dependent lysyl-phosphatidylglycerol synthase (L-PGS) enables bacteria to cope with cationic peptides that are harmful to the integrity of the cell membrane. Accordingly, these synthases also have been designated as multiple peptide resistance factors (MprF). They consist of a separable C-terminal catalytic domain and an N-terminal transmembrane flippase domain. Here we present the X-ray crystallographic structure of the catalytic domain of A-PGS from the opportunistic human pathogen Pseudomonas aeruginosa. In parallel, the structure of the related lysyl-phosphatidylglycerol-specific L-PGS domain from Bacillus licheniformis in complex with the substrate analog L-lysine amide is presented. Both proteins reveal a continuous tunnel that allows the hydrophobic lipid substrate PG and the polar aminoacyl-tRNA substrate to access the catalytic site from opposite directions. Substrate recognition of A-PGS versus L-PGS was investigated using misacylated tRNA variants. The structural work presented here in combination with biochemical experiments using artificial tRNA or artificial lipid substrates reveals the tRNA acceptor stem, the aminoacyl moiety, and the polar head group of PG as the main determinants for substrate recognition. A mutagenesis approach yielded the complementary amino acid determinants of tRNA interaction. These results have broad implications for the design of L-PGS and A-PGS inhibitors that could render microbial pathogens more susceptible to antimicrobial compounds.

Functional Class I and II Amino Acid-activating Enzymes Can Be Coded by Opposite Strands of the Same Gene.

Aminoacyl-tRNA synthetases (aaRS) catalyze both chemical steps that translate the universal genetic code. Rodin and Ohno offered an explanation for the existence of two aaRS classes, observing that codons for the most highly conserved Class I active-site residues are anticodons for corresponding Class II active-site residues. They proposed that the two classes arose simultaneously, by translation of opposite strands from the same gene. We have characterized wild-type 46-residue peptides containing ATP-binding sites of Class I and II synthetases and those coded by a gene designed by Rosetta to encode the corresponding peptides on opposite strands. Catalysis by WT and designed peptides is saturable, and the designed peptides are sensitive to active-site residue mutation. All have comparable apparent second-order rate constants 2.9-7.0E-3 M(-1) s(-1) or ∼750,000-1,300,000 times the uncatalyzed rate. The activities of the two complementary peptides demonstrate that the unique information in a gene can have two functional interpretations, one from each complementary strand. The peptides contain phylogenetic signatures of longer, more sophisticated catalysts we call Urzymes and are short enough to bridge the gap between them and simpler uncoded peptides. Thus, they directly substantiate the sense/antisense coding ancestry of Class I and II aaRS. Furthermore, designed 46-mers achieve similar catalytic proficiency to wild-type 46-mers by significant increases in both kcat and Km values, supporting suggestions that the earliest peptide catalysts activated ATP for biosynthetic purposes.

3,5-Dicaffeoylquinic acid isolated from Artemisia argyi and its ester derivatives exert anti-leucyl-tRNA synthetase of Giardia lamblia (GlLeuRS) and potential anti-giardial effects.

An aqueous ethanol extract of Artemisia argyi inhibited the aminoacylation activity of LeuRS from Giardia lamblia (GlLeuRS). The bioassay-guided fractionation of the extract led to the isolation of 3,5-dicaffeoylquinic acid (1), with an IC50 of 5.82 µg/mL. The ester derivatives of 1 were also found to possess strong anti-GlLeuRS effects, with IC50 values of 1.79, 5.51 and 2.56 µg/mL respectively. Anti-giardial assay showed that the derivatives, especially 3,5-dicaffeoylquinic acid propyl ester (4) (IC50=4.62 µg/mL), were effective at killing G. lamblia.

A horizontally transferred tRNA(Cys) gene in the sugar beet mitochondrial genome: evidence that the gene is present in diverse angiosperms and its transcript is aminoacylated.

Of the two tRNA(Cys) (GCA) genes, trnC1-GCA and trnC2-GCA, previously identified in mitochondrial genome of sugar beet, the former is a native gene and probably a pseudo-copy, whereas the latter, of unknown origin, is transcribed into a tRNA [tRNA(Cys2) (GCA)]. In this study, the trnC2-GCA sequence was mined from various public databases. To evaluate whether or not the trnC2-GCA sequence is located in the mitochondrial genome, the relative copy number of its sequence to nuclear gene was assessed in a number of angiosperm species, using a quantitative real-time PCR assay. The trnC2-GCA sequence was found to exist sporadically in the mitochondrial genomes of a wide range of angiosperms. The mitochondrial tRNA(Cys2) (GCA) species from sugar beet (Beta vulgaris), spinach (Spinacea oleracea) and cucumber (Cucumis sativus) were found to be aminoacylated, indicating that they may participate in translation. We also identified a sugar beet nuclear gene that encodes cysteinyl-tRNA synthetase, which is dual-targeted to mitochondria and plastids, and may aminoacylate tRNA(Cys2) (GCA). What is of particular interest is that trnC1-GCA and trnC2-GCA co-exist in the mitochondrial genomes of eight diverse angiosperms, including spinach, and that the spinach tRNA(Cys1) (GCA) is also aminoacylated. Taken together, our observations lead us to surmise that trnC2-GCA may have been horizontally transferred to a common ancestor of eudicots, followed by co-existence and dual expression of trnC1-GCA and trnC2-GCA in mitochondria with occasional loss or inactivation of either trnC-GCA gene during evolution.

Molecular and cellular specificity of post-translational aminoacyl isomerization in the crustacean hyperglycaemic hormone family.

D-aminoacyl residues have been detected in various animal peptides from several taxa, especially vertebrates and arthropods. This unusual polymorphism was shown to occur in isoforms of the crustacean hyperglycaemic hormone (CHH) of the American lobster because a D-phenylalanyl residue was found in position 3 of the sequence (CHH and D-Phe3 CHH). In the present study, we report the detailed strategy used to characterize, in the lobster neuroendocrine system, isomers of another member of the CHH family, vitellogenesis inhibiting hormone (VIH). We have demonstrated that the fourth residue is either an L- or a D- tryptophanyl residue (VIH and D-Trp4 VIH). Furthermore, use of antisera specifically recognizing the epimers of CHH and VIH reveals that aminoacyl isomerization occurs in specialized cells of the X organ-sinus gland neurosecretory system and that the D-forms of the two neuropeptides are not only present in the same cells, but, importantly, also are co-packaged within the same secretory vesicles.

Ajo y riesgo cardiovascular / Garlic and cardiovascular risk

El ajo es una de las plantas curativas más antiguas de la que tenemos referencia. En el presente trabajo, revisaremos las evidencias existentes sobre el consumo de ajo y el riesgo cardiovascular, analizando su papel sobre el perfil lipídico, el endotelio vascular y la agregación plaquetaria. El consumo regular de 5 g de ajo crudo dos veces al día durante 42 días disminuye los niveles de colesterol total y triglicéridos, no obstante la mayor parte de los estudios realizados en el campo de los lípidos han sido llevados a cabo en animales. En modelos animales (perros y ratas) se ha demostrado los efectos hipotensores del ajo de manera dosis dependiente. Algunos trabajos han demostrado que el ajo activa la fibrinolisis, además de suprimir el sistema de coagulación previniendo así la formación de trombos. Estos resultados se han corroborado en otro trabajo, en el cual se demuestra que el extracto alcohólico de ajo es un potente inhibidor de la agregación plaquetaria. En resumen, los efectos del ajo sobre la disminución del riesgo cardiovascular son importantes. No obstante los estudios en humanos son escasos y todavía falta por demostrar que estos efectos disminuyan los eventos cardiovasculares así como la cantidad necesaria de ajo para obtener estos beneficios

Garlic is one of the oldest dietary vegetables. In this article, we´ll review the evidences of cardiovascular risk factors and garlic consumption, analizing the influence on lipid levels, vascular endothelium and platelet aggregation. Regular consumption of 5 g (raw garlic) twice per day during 42 days decreases levels of cholesterol and triglyceride. However, almost all lipid studies have been realized in animals. In animal models (dogs and rats), it has been demonstrated a dose depending hypotensive effect, too. Garlic consumption has demonstrated an activation of fibrinolisis and a down regulation of coagulation, with a decrease in the rate of thrombogenesis. These results have been repeated by otherwork, in which a garlic alcoholic extract is a potent inhibitor of platelet aggregation. In summary, the effects of garlic on cardiovascular risk are important. However, it is necessary to demonstrate a decrease on cardiovascular events and to elucidate the amount of garlic to obtain benefict effects, most studies are necessary

Solid-phase combinatorial synthesis of a lysyl-tRNA synthetase (LysRS) inhibitory library.

The solid-phase combinatorial synthesis of a new library with potential inhibitory activity against the cytoplasmic lysyl-tRNA synthetase (LysRS) isoform of Trypanosoma brucei is described. The library has been specifically designed to mimic the lysyl adenylate complex. The design was carried out by dividing the complex into four modular parts. Proline derivatives (cis-gamma-amino-L-proline or trans-gamma-hydroxy-L-proline) were chosen as central scaffolds. After primary screening, three compounds of the library caused in vitro inhibition of the tRNA aminoacylation reaction in the low micromolar range.

Human tryptophanyl-tRNA synthetase binds with heme to enhance its aminoacylation activity.

Mammalian tryptophanyl-tRNA synthetases (TrpRSs) are Zn2+-binding proteins that catalyze the aminoacylation of tRNATrp. The cellular expression level of human TrpRS is highly upregulated by interferon-gamma (IFN-gamma). In this study, a heme biosynthesis inhibitor, succinylacetone (SA), was found to inhibit cellular TrpRS activity in IFN-gamma-activated cells without affecting TrpRS protein expression. In addition, supplementation of lysates from the SA-treated cells with hemin fully restored TrpRS activity to control levels. Biochemical analyses using purified TrpRS demonstrated that heme can interact strongly with Zn2+-depleted human full-length TrpRS with a stoichiometric heme:protein ratio of 1:1 to enhance the aminoacylation activity significantly. In contrast, the Zn2+-bound form of TrpRS did not bind heme. Further studies using site-directed mutagenesis clarified that the Zn2+-unbound human H130R mutant cannot bind heme. These results provide the first evidence of the involvement of heme in regulation of TrpRS aminoacylation activity. The regulation mechanism and its physiological roles are discussed.

Crystal structure of tryptophanyl-tRNA synthetase complexed with adenosine-5' tetraphosphate: evidence for distributed use of catalytic binding energy in amino acid activation by class I aminoacyl-tRNA synthetases.

Tryptophanyl-tRNA synthetase (TrpRS) is a functionally dimeric ligase, which specifically couples hydrolysis of ATP to AMP and pyrophosphate to the formation of an ester bond between tryptophan and the cognate tRNA. TrpRS from Bacillus stearothermophilus binds the ATP analogue, adenosine-5' tetraphosphate (AQP) competitively with ATP during pyrophosphate exchange. Estimates of binding affinity from this competitive inhibition and from isothermal titration calorimetry show that AQP binds 200 times more tightly than ATP both under conditions of induced-fit, where binding is coupled to an unfavorable conformational change, and under exchange conditions, where there is no conformational change. These binding data provide an indirect experimental measurement of +3.0 kcal/mol for the conformational free energy change associated with induced-fit assembly of the active site. Thermodynamic parameters derived from the calorimetry reveal very modest enthalpic changes, consistent with binding driven largely by a favorable entropy change. The 2.5 A structure of the TrpRS:AQP complex, determined de novo by X-ray crystallography, resembles that of the previously described, pre-transition state TrpRS:ATP complexes. The anticodon-binding domain untwists relative to the Rossmann-fold domain by 20% of the way toward the orientation observed for the Products complex. An unexpected tetraphosphate conformation allows the gamma and deltad phosphate groups to occupy positions equivalent to those occupied by the beta and gamma phosphates of ATP. The beta-phosphate effects a 1.11 A extension that relocates the alpha-phosphate toward the tryptophan carboxylate while the PPi mimic moves deeper into the KMSKS loop. This configuration improves interactions between enzyme and nucleotide significantly and uniformly in the adenosine and PPi binding subsites. A new hydrogen bond forms between S194 from the class I KMSKS signature sequence and the PPi mimic. These complementary thermodynamic and structural data are all consistent with the conclusion that the tetraphosphate mimics a transition-state in which the KMSKS loop develops increasingly tight bonds to the PPi leaving group, weakening linkage to the Palpha as it is relocated by an energetically favorable domain movement. Consistent with extensive mutational data on Tyrosyl-tRNA synthetase, this aspect of the mechanism develops high transition-state affinity for the adenosine and pyrophosphate moieties, which move significantly, relative to one another, during the catalytic step.

Seven new aminoacyl sugars in Ipomoea batatas.

An analysis of the polar extracts from sweet potato Ipomoea batatas (Convolvulaceae) led to the isolation of seven unknown aminoacyl sugars. On the basis of 1D, 2D NMR, and mass spectrometry data, the structures of the compounds were elucidated as: beta-D-fructofuranosyl-(2 --> 1)-alpha-D-[2-O-valyl]-glucopyranoside (1), beta-D-fructofuranosyl-(2 --> 1)-alpha-D-[2-O-tyrosyl]-glucopyranoside (2), beta-D-fructofuranosyl-(2 --> 1)-alpha-D-[2-O-threonyl]-glucopyranoside (3), beta-D-fructofuranosyl-(2 --> 1)-alpha-D-[2-O-hystidyl]-glucopyranoside (4), 2-beta-D-fructofuranosyl-(2 --> 1)-alpha-D-[2-O-alanyl]-glucopyranoside (5), beta-D-fructofuranosyl-(2 --> 1)-alpha-D-[2-O-tryptophanyl]-glucopyranoside (6), and beta-D-fructofuranosyl-(2 --> 1)-alpha-D-[2-O-glycyl]-glucopyranoside (7).

Inhibition of peptide chain initiation in lysates from ATP-depleted cells.I. Stages in the evolution of the lesion and its reversal by thiol compounds, cyclic AMP or purine derivatives and phosphorylated sugars.

Anaerobic incubation of rabbit reticulocytes at 37 degrees C in Krebs-Ringer solution supplemented with hemin but devoid of glucose resulted at the end of 1-2h in a drastic decline of their ATP content and an attendant arrest of protein synthesis. Subsequent provision of glucose and reoxygenation of the cells was followed by a rapid replenishment of the ATP pool, while resumption of protein synthesis was markedly delayed. This lag period could be considerably reduced by addition of 5-10 mM adenine or 2,6-diaminopurine to the incubation medium. Lysates prepared from ATP-depleted cells exhibited disaggregation of the polysomes and an inhibition of the nedogenously coded protein synthesis, when tested in a cell-free system supplied with an adequate ATP generator. Both alterations increased in severity with the progressive decay of the intracellular ATP pool. The early phase of partial inhibition following a 40-70% decrease of the cellular ATP level was fully reversible by fortifying the cell-free preparation with dithiothreitol or a suitable NADPH-generating system. Aternative, the inhibition could be also overcome by millimolar amounts of adenine, 2,6-diaminopurine and a variety of other purine derivatives or cyclic AMP. The effect of these compounds was unrelated to the endogenous cyclic AMP pool. Joint addition of both dithiothreitol and cyclic AMP or adenine was necessary for relieving the initiation block in lysates derived from cells depleted of 80-90% of their ATP content. On further aggravating the conditions of energy starvation, an additional requirement for phosphorylated sugars, e.g. glucose 6-phosphate or fructose 1,6-diphosphate, became apparent. ATP depletion brought about by exposing the cells to Antimycin A or 2,4-dinitrophenol resulted in a lesion which was indistinguishable from that induced by anaerobic incubation. On the other hand, energy deprivation in cell-free lysates from untreated reticulocytes, preincubated in the absence of an ATP-generating system failed to duplicate the deleterious effect of intracellular ATP depletion. Some aspects bearing on the biochemical mechanism of the lesion and its reversal are discussed in the light of the available data.