Acetylated Amino Acids Research Articles

Systemic characteristics of biomarkers and differential metabolites of raw and ripened pu-erh teas by chemical methods combined with a UPLC-QQQ-MS-based metabolomic approach

Pu-erh tea is one of the most popular beverages in China and Southeast Asia, however, influences from fermentation and storage on its chemical profile and quality are unclear. Thus, bioactivities and metabolomes of raw (17-raw) and ripened teas (17-rip through 06-rip) were assessed using chemical methods and a UPLC-QQQ-MS-based metabolomic approach. Results evidence that chemical components and antioxidant activities of 17-rip through 06-rip were similar but lower than those of 17-raw. Subsequently, 842 metabolites were identified from 17-raw, 17-rip and 06-rip, of which 20 and 19 metabolites were biomarkers for discerning 17-rip from 17-raw and 06-rip, respectively. Between 17-raw and 17-rip, 536 differential metabolites were identified, and 17-rip contained higher levels of gallic acid, acetyl amino acids, purine alkaloids, pyrimidine alkaloids and non-glycoside flavonoids and lower levels of sour compounds, quinic acid derivatives, amino acids, flavonoids glycosides, and flavan-3-ols; all of them were found to be positively but gallic acid and acetyl amino acids negatively correlated with two tested bioactivities. Between 17-rip and 06-rip, 175 differential metabolites were identified, among which alkaloids positively correlated to the two bioactivities. Overall, this study identified biomarkers distinguishing teas with different fermentation processes and storage times and different metabolites affecting their tastes and bioactivities.

Urine metabolomic analysis for monitoring internal load in professional football players.

The design of training programs for football players is not straightforward due to intra- and inter-individual variability that leads to different physiological responses under similar training loads. To study the association between the external load, defined by variables obtained using electronic performance tracking systems (EPTS), and the urinary metabolome as a surrogate of the metabolic adaptation to training. Urine metabolic and EPTS data from 80 professional football players collected in an observational longitudinal study were analyzed by ultra-performance liquid chromatography coupled to electrospray ionization quadrupole time-of-flight mass spectrometry and assessed by partial least squares (PLS) regression. PLS models identified steroid hormone metabolites, hypoxanthine metabolites, acetylated amino acids, intermediates in phenylalanine metabolism, tyrosine, tryptophan metabolites, and riboflavin among the most relevant variables associated with external load. Metabolic network analysis identified enriched pathways including steroid hormone biosynthesis and metabolism of tyrosine and tryptophan. The ratio of players showing a deviation from the PLS model of adaptation to exercise was higher among those who suffered a muscular lesion compared to those who did not. There was a significant association between the external load and the urinary metabolic profile, with alteration of biochemical pathways associated with long-term adaptation to training. Future studies should focus on the validation of these findings and the development of metabolic models to identify professional football players at risk of developing muscular injuries.

Characterization of thermostable aminoacylase from Geobacillus sp. strain SZN

Aminoacylase (EC 3.5.1.14) hydrolyzes N–acetylated amino acids to produce amino acids. Although thermostable aminoacylase has been commercially produced since 2004, there was a knowledge gap in the field of understanding aminoacylase thermostability from a structural point of view. This study investigated the physical and structural properties of the purified thermostable aminoacylase SZN. The spectropolarimetry data for structural determination has indicated a gradual decrease of α-helix from 36 to 27.6%, followed by tremendous disorientation of the structure at the transition of temperatures from 60 to 70°C (27.6 to 19.5%). In contrast, the percentage of β-sheet has increased steadily over the tested temperatures. The α-helix, where notable metal binding and catalytic residues are located, was totally weakened at temperatures above 70C, thus resulted in loss of activity. The loss of the α-helical structure could further explain drastic deterioration of activity at temperatures beyond 70C. The activity of aminoacylase SZN was enhanced by divalent metal ions, such as Mn2+ and Cu2+, and inhibited by detergent Triton-X-100. As a conclusion, the isolated aminoacylase SZN was characterized as a thermostable enzyme based on the α-helical structure integrity and functional stability in high temperatures. This enzyme could be used as an alternative enzyme for bioindustries in view of its activity enhancement in high temperatures and stability in various tested inhibitors.

A non-derivatized method for simultaneous quantitation of proteinogenic, urea-cycle, and acetylated amino acids by liquid chromatography–high-resolution mass spectrometry

The quantification of amino acids as freely dissolved compounds or from hydrolyzed peptides and proteins is a common endeavor in biomedical and environmental sciences. In order to avoid the drawbacks of derivatization and application challenges of tandem mass spectrometry, we present here a robust 13-min liquid chromatography coupled with a full-scan mass spectrometry method to achieve rapid detection and quantification of 30 amino acids without derivatization. We combined hydrophilic interaction liquid chromatography with heated electrospray ionization and high-resolution mass spectrometry operated with polarity switching to analyze the 20 proteinogenic amino acids, ornithine, citrulline, homoserine, cystine, and six acetylated amino acids. We obtained high mass accuracy and good precision of the targeted amino acids. Limits of detection ranged from 0.017 to 1.3 µM. Noteworthy for environmental samples, we found comparable ionization efficiency and quantitative detection for the majority of the analytes prepared with pure water versus a high-salt solution. We applied the method to profile carbon source-dependent secretions of amino acids by Pseudomonas protegens Pf-5, a well-known plant-beneficial bacterium.

Detecting acetylated aminoacids in blood serum using hyperpolarized 13C-1Η-2D-NMR.

Dynamic Nuclear Polarization (DNP) can substantially enhance the sensitivity of NMR experiments. Among the implementations of DNP, ex-situ dissolution DNP (dDNP) achieves high signal enhancement levels owing to a combination of a large temperature factor between 1.4 and 300 K with the actual DNP effect in the solid state at 1.4 K. For sufficiently long T1 relaxation times much of the polarization can be preserved during dissolution with hot solvent, thus enabling fast experiments during the life time of the polarization. Unfortunately, for many metabolites found in biological samples such as blood, relaxation times are too short to achieve a significant enhancement. We have therefore introduced 13C-carbonyl labeled acetyl groups as probes into amino acid metabolites using a simple reaction protocol. The advantage of such tags is a sufficiently long T1 relaxation time, the possibility to enhance signal intensity by introducing 13C, and the possibility to identify tagged metabolites in NMR spectra. We demonstrate feasibility for mixtures of amino acids and for blood serum. In two-dimensional dDNP-enhanced HMQC experiments of these samples acquired in 8 s we can identify acetylated amino acids and other metabolites based on small differences in chemical shifts.

1867-P: The Role of Ketone Bodies in Diabetes Pathogenesis Using Untargeted Metabolomics

The pathogenesis of human T2D is linked to the progression of nonalcoholic fatty liver disease and steatohepatitis (NAFLD/NASH). In the normal fed state, impairments of hepatocyte ketogenesis and Kupffer cell mitochondrial metabolism of ketone bodies conspire to drive hepatic injury, and lobular fibrosis - key features increasing insulin resistance and T2D. Preliminary data suggest that a local circuit of intrahepatic ketone metabolism may protect the liver from lobular injury, which involves distribution of ketone body carbon into numerous metabolic pathways. Nonetheless, the metabolic products of local hepatic ketone body metabolism that confer protection to the liver, and which are therefore linked to insulin resistance and T2D, remain unknown. Here, we use stable isotope tracing untargeted metabolomics (ITUM) to reveal the chemical space penetrated in vivo by D-[13C]β-hydroxybutyrate (D-βOHB) and its oxidized redox partner [13C]acetoacetate (AcAc). A kinetic study revealed rapid clearance (∼30 min) of both ketones from the bloodstream when introduced as intraperitoneal boluses into wild type mice. Many 13C-labeled metabolites were tissue-specific, particularly distinctly acetylated amino acids. Labeling of TCA cycle intermediates by 13C-labeled ketone bodies also varied by tissue, and by the specific labeled ketone body delivered. Finally, recurrent administration of unlabeled AcAc protected mice from high fat, fibrogenic diet-induced hepatic injury and fibrosis, while unlabeled D-βOHB exacerbated injury and fibrosis. In conclusion, the two ketone bodies AcAc and D-βOHB, strikingly exhibit distinct metabolic and phenotypic effects in the context of NAFLD/NASH, and insulin resistance. Disclosure P. Puchalska: None. J.E. Lengfeld: None. D.B. Stagg: None. P.A. Crawford: Consultant; Spouse/Partner; Medtronic.

Abstract B66: BET inhibition remodels tumor stroma and suppresses progression of human pancreatic cancer

Abstract Background and Aims: Pancreatic ductal adenocarcinoma (PDAC) is well characterized by dense fibrotic stroma with abundant cancer-associated fibroblasts (CAFs). As CAFs are activated during tumorigenesis and acquire tumor-promoting properties, activated CAFs have been implicated in PDAC progression; however, the precise mechanisms of their activation remain largely unknown. The bromodomain and extraterminal (BET) domain proteins are epigenetic reader proteins that recognize acetylated amino acid residues on histone tails and facilitate gene transcription. Recent studies have demonstrated therapeutic efficacy of BET inhibitors on various cancers including PDAC, mainly through suppression of c-myc transcription; however, how BET inhibitors suppress PDAC growth and their effects on CAFs remains largely unknown. Using patient-derived tumor xenografts (PDX) and primary CAFs, we investigated the therapeutic efficacy and dissected the underlying mechanisms of a BET inhibitor, JQ1, on human PDAC and CAFs. Methods: We established PDX lines and primary CAFs from surgically resected human PDAC specimen. For in vivo analyses, mice bearing subcutaneous tumor were treated with vehicle or JQ1. For in vitro analyses, patient-derived PDAC cells and CAFs were treated with vehicle or JQ1 and analyzed separately. To explore the pro-tumorigenic role of secretion from CAFs, PDAC cells were cultured with conditioned medium (CM) that was collected from DMSO- or JQ1- treated CAFs. Chromatin immunoprecipitation (ChIP) assay was performed to assess the binding of transcription factors and histone modifications which are associated with altered gene expression in CAFs by JQ1 treatment. Results: In vivo experiments revealed that volumes and weights of subcutaneous PDX tumors were significantly smaller in JQ1-treated mice than vehicle-treated mice. Unexpectedly, however, JQ1 exerted only minimal effects to the proliferation of PDAC cells that were isolated from PDX tumors and cultured in vitro, suggesting the involvement of cell-extrinsic mechanisms in the JQ1-mediated suppression of tumor growth in vivo. Of note, histopathological analysis of PDX tumors revealed that JQ1 treatment dramatically ameliorated desmoplastic change, with reduction in extracellular matrix (ECM) deposition and α-SMA expressing CAFs. As α-SMA expression and ECM production is a hallmark of activated CAFs, we hypothesized that JQ1 might inactivate CAFs, thereby reducing their tumor-promoting properties. To test this hypothesis, qPCR was performed to analyze gene expression in primary CAFs cultured in vitro and also in stromal cells in PDX tumors in vivo. As expectedly, JQ1 suppressed the expression of genes implicated in the properties of activated CAF, including ECM, cytokines and growth factors both in vitro and in vivo. Furthermore, when PDAC cells were cultured with CM from DMSO–treated CAFs, proliferation of PDAC cells were promoted along with activation of MAPK, AKT, and STAT3 pathways, which was abrogated when cultured with CM from JQ1-treated CAFs. Consistently, immunoblotting and immunohistochemistry of PDX tumors demonstrated that JQ1 reduced phosphorylation of ERK, AKT, and STAT3 in PDAC cells in vivo. Mechanistically, we found that JQ1 suppressed hedgehog and TGF-β/SMAD3 pathways, both of which play central roles in CAF activation, through disruption of BRD4 recruitment to the promoter regions of their target genes. Conclusions: BET proteins are critical regulators of CAF-activation in PDAC. Inactivation of CAFs by BET inhibition offers a novel therapeutic approach for PDAC. Citation Format: Keisuke Yamamoto, Keisuke Tateishi, Yotaro Kudo, Mayumi Hoshikawa, Mariko Tanaka, Takuma Nakatsuka, Hiroaki Fujiwara, Koji Miyabayashi, Ryota Takahashi, Yasuo Tanaka, Hideaki Ijichi, Yousuke Nakai, Hiroyuki Isayama, Yasuyuki Morishita, Taku Aoki, Yoshihiro Sakamoto, Kiyoshi Hasegawa, Norihiro Kokudo, Masashi Fukayama, Kazuhiko Koike.{Authors}. BET inhibition remodels tumor stroma and suppresses progression of human pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr B66.

Probing the metabolic network in bloodstream-form Trypanosoma brucei using untargeted metabolomics with stable isotope labelled glucose.

Metabolomics coupled with heavy-atom isotope-labelled glucose has been used to probe the metabolic pathways active in cultured bloodstream form trypomastigotes of Trypanosoma brucei, a parasite responsible for human African trypanosomiasis. Glucose enters many branches of metabolism beyond glycolysis, which has been widely held to be the sole route of glucose metabolism. Whilst pyruvate is the major end-product of glucose catabolism, its transamination product, alanine, is also produced in significant quantities. The oxidative branch of the pentose phosphate pathway is operative, although the non-oxidative branch is not. Ribose 5-phosphate generated through this pathway distributes widely into nucleotide synthesis and other branches of metabolism. Acetate, derived from glucose, is found associated with a range of acetylated amino acids and, to a lesser extent, fatty acids; while labelled glycerol is found in many glycerophospholipids. Glucose also enters inositol and several sugar nucleotides that serve as precursors to macromolecule biosynthesis. Although a Krebs cycle is not operative, malate, fumarate and succinate, primarily labelled in three carbons, were present, indicating an origin from phosphoenolpyruvate via oxaloacetate. Interestingly, the enzyme responsible for conversion of phosphoenolpyruvate to oxaloacetate, phosphoenolpyruvate carboxykinase, was shown to be essential to the bloodstream form trypanosomes, as demonstrated by the lethal phenotype induced by RNAi-mediated downregulation of its expression. In addition, glucose derivatives enter pyrimidine biosynthesis via oxaloacetate as a precursor to aspartate and orotate.

Continuous colorimetric assay that enables high-throughput screening of N-acetylamino acid racemases.

N-Acetyl amino acid racemases (NAAARs) have demonstrated their potential in the enzymatic synthesis of chiral amino acids, molecules of significant biotechnology interest. In order to identify novel activities and to improve these enzymes by engineering approaches, suitable screening methods are necessary. Previous engineering of the NAAAR from Amycolatopsis Ts-1-60 was achieved by relying on an in vivo selection system that linked the viability of an E. coli L-methionine auxotroph to the activity of the improved enzyme. However, this assay was only suitable for the screening of N-acetyl-D-methionine, therefore limiting the potential to evolve this enzyme toward other natural or non-natural acetylated amino acids. Here, we report the optimization and application of a spectrophotometric microtiter-plate-based assay for NAAAR. The assay is based on the detection of the amino acid reaction product formed by hydrolysis of the N-acylated substrate by an L-amino acid acylase and its subsequent oxidation by an FAD-dependent L-amino acid oxidase (L-AAO). Cofactor recycling of the L-AAO leads to the formation of hydrogen peroxide which is easily monitored using horseradish peroxidase (HRP) and o-dianisidine. This method allowed for the determination of the kinetic parameters of NAAAR and led to the identification of N-acetyl-D-naphthylalanine as a novel NAAAR substrate. This robust method is also suitable for the high-throughput screening of NAAAR mutant gene libraries directly from cell lysates.

Unexpected similarities between the Schizosaccharomyces and human blood metabolomes, and novel human metabolites.

Metabolomics, a modern branch of chemical biology, provides qualitative and quantitative information about the metabolic states of organisms or cells at the molecular level. Here we report non-targeted, metabolomic analyses of human blood, using liquid chromatography-mass spectrometry (LC-MS). We compared the blood metabolome to the previously reported metabolome of the fission yeast, Schizosaccharomyces pombe. The two metabolomic datasets were highly similar: 101 of 133 compounds identified in human blood (75%) were also present in S. pombe, and 45 of 57 compounds enriched in red blood cells (RBCs) (78%) were also present in yeast. The most abundant metabolites were ATP, glutathione, and glutamine. Apart from these three, the next most abundant metabolites were also involved in energy metabolism, anti-oxidation, and amino acid metabolism. We identified fourteen new blood compounds, eight of which were enriched in RBCs: citramalate, GDP-glucose, trimethyl-histidine, trimethyl-phenylalanine, trimethyl-tryptophan, trimethyl-tyrosine, UDP-acetyl-glucosamine, UDP-glucuronate, dimethyl-lysine, glutamate methyl ester, N-acetyl-(iso)leucine, N-acetyl-glutamate, N2-acetyl-lysine, and N6-acetyl-lysine. Ten of the newly identified blood metabolites were also detected in S. pombe, and ten of the 14 newly identified blood metabolites were methylated or acetylated amino acids. Trimethylated or acetylated free amino acids were also abundant in white blood cells. It may be possible to investigate their physiological roles using yeast genetics.

Prediction of glycoprotein secondary structure using ATR-FTIR

Glycoproteins are important biomolecules with a diverse array of structural and signaling functions in biology. Determination of glycoprotein secondary structure is becoming increasingly important in aiding the understanding of how these molecules function in biological environments and disease. Furthermore, glycoproteins such as mucins are being evaluated in various nano-engineering processes that require knowledge of how the underlying secondary structure might alter in different target environments. We have developed an analytical procedure for predicting the secondary structures of glycoprotein using ATR-FTIR on dry film. Using Bovine submaxillary mucin (BSM) as a glycoprotein model, we determined the additive infrared spectral pattern of acetyl amino sugars and amino acids that could contribute to the absorbance in the Amide I band of BSM through empirical data. We show through subtraction of these spectra how the absorbance pattern of the protein backbone can be determined in order to predict glycoprotein secondary structure. The analysis predicted a predominant pattern of random coil, beta sheet and beta turn secondary structure for BSM after carbohydrate and amino acid spectral subtraction in agreement with other methods. Our relatively simple approach can be applied to predict secondary structure in other glycoproteins.

Comparative Metabolomics in Glycine max and Glycine soja under Salt Stress To Reveal the Phenotypes of Their Offspring

Metabolomics is developing as an important functional genomics tool for understanding plant systems' response to genetic and environmental changes. Here, we characterized the metabolic changes of cultivated soybean C08 (Glycine max L. Merr) and wild soybean W05 (Glycine soja Sieb.et Zucc.) under salt stress using MS-based metabolomics, in order to reveal the phenotypes of their eight hybrid offspring (9H0086, 9H0124, 9H0391, 9H0736, 9H0380, 9H0400, 9H0434, and 9H0590). Total small molecule extracts of soybean seedling leaves were profiled by gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-Fourier transform mass spectrometry (LC-FT/MS). We found that wild soybean contained higher amounts of disaccharides, sugar alcohols, and acetylated amino acids than cultivated soybean, but with lower amounts of monosaccharides, carboxylic acids, and unsaturated fatty acids. Further investigations demonstrated that the ability of soybean to tolerate salt was mainly based on synthesis of compatible solutes, induction of reactive oxygen species (ROS) scavengers, cell membrane modifications, and induction of plant hormones. On the basis of metabolic phenotype, the salt-tolerance abilities of 9H0086, 9H0124, 9H0391, 9H0736, 9H0380, 9H0400, 9H0434, and 9H0590 were discriminated. Our results demonstrated that MS-based metabolomics provides a fast and powerful approach to discriminate the salt-tolerance characteristics of soybeans.

On the transferability of fractional contributions to the hydration free energy of amino acids

This study reports the application of the quantum mechanical self-consistent reaction field MST method to compute the solvation profile in water of the twenty natural amino acids. The aim is to derive intrinsic fractional contributions to the hydration free energy and to examine their transferability to peptides. To this end, IEF-MST calculations have been performed at the B3LYP/6-31G(d) level for the series of acetyl amino acid amides, which were chosen as model compounds. In order to account for the flexibility of both the backbone and the side chain in deriving the hydration fractional contributions, calculations have been performed for representative conformers taken from the Dunbrack’s backbone-dependent conformational library. The results allow us to dissect the hydration free energy into backbone and side chain contributions and examine the conformational dependence of the fragmental contributions to hydration. For the backbone, different hydration contributions are found for α-helical and β-sheet conformations, which mainly reflect differences in the electrostatic contribution to hydration of the carbonyl group. In contrast, the conformational flexibility of the side chain is found to have little impact on the fractional contribution to hydration. These findings should be valuable to refine semiempirical methods for predicting solvation properties of peptides and proteins in large-scale genomic studies.

Structural and Energetic Effects in the Molecular Recognition of Acetylated Amino Acids by 18-Crown-6

Absolute 18-crown-6 (18C6) binding affinities of four protonated acetylated amino acids (AcAAs) are determined using guided ion beam tandem mass spectrometry techniques. The AcAAs examined in this work include: N-terminal acetylated lysine (N(α)-AcLys), histidine (N(α)-AcHis), and arginine (N(α)-AcArg) as well as side chain acetylated lysine (N(ε)-AcLys). The kinetic-energy-dependent cross sections for collision-induced dissociation (CID) of the (AcAA)H(+)(18C6) complexes are analyzed using an empirical threshold law to extract absolute 0 and 298 K (AcAA)H(+)-18C6 bond dissociation energies (BDEs) after accounting for the effects of multiple collisions, kinetic and internal energy distributions of the reactants, and unimolecular dissociation lifetimes. Theoretical electronic structure calculations are performed to determine stable geometries and energetics for neutral and protonated 18C6 and the AcAAs as well as the proton bound complexes of these species, (AcAA)H(+)(18C6), at the B3LYP/6-311+G(2d,2p)//B3LYP/6-31 G* and M06/6-311+G(2d,2p)//B3LYP/6-31G* levels of theory. For all four (AcAA)H(+)(18C6) complexes, loss of neutral 18C6 corresponds to the most favorable dissociation pathway. At elevated energies, products arising from sequential dissociation of the primary CID product, H(+)(AcAA), are also observed. Protonated N(α)-AcLys exhibits a greater 18C6 binding affinity than other protonated N(α)-AcAAs, suggesting that the side chains of Lys residues are the preferred binding sites for 18C6 complexation to peptides and proteins. N(α)-AcLys exhibits a greater 18C6 binding affinity than N(ε)-AcLys, suggesting that binding of 18C6 to the side chain of Lys residues is more favorable than to the N-terminal amino group of Lys.

Identification and Characterisation of a Novel Acylpeptide Hydrolase from Sulfolobus Solfataricus: Structural and Functional Insights

A novel acylpeptide hydrolase, named APEH-3Ss, was isolated from the hypertermophilic archaeon Sulfolobus solfataricus. APEH is a member of the prolyl oligopeptidase family which catalyzes the removal of acetylated amino acid residues from the N terminus of oligopeptides. The purified enzyme shows a homotrimeric structure, unique among the associate partners of the APEH cluster and, in contrast to the archaeal APEHs which show both exo/endo peptidase activities, it appears to be a “true” aminopeptidase as exemplified by its mammalian counterparts, with which it shares a similar substrate specificity. Furthermore, a comparative study on the regulation of apeh gene expression, revealed a significant but divergent alteration in the expression pattern of apeh-3Ss and apehSs (the gene encoding the previously identified APEHSs from S. solfataricus), which is induced in response to various stressful growth conditions. Hence, both APEH enzymes can be defined as stress-regulated proteins which play a complementary role in enabling the survival of S. solfataricus cells under different conditions. These results provide new structural and functional insights into S. solfataricus APEH, offering a possible explanation for the multiplicity of this enzyme in Archaea.

Comparison of the Conformational Behavior of Amino Acids and N-Acetylated Amino Acids: A Theoretical and Matrix-Isolation FT-IR Study of N-Acetylglycine

Within the structure determination task for peptides, which is of large interest due to the relation between structure and functionality, infrared spectra can provide detailed information on the conformational behavior. The conformational landscape ofN-acetylgycine has been studied by a combined theoretical and matrix-isolation FT-IR study. The acetylation simulates an amino acid a peptide bond. Four stable conformations were found at the MP2/6-31++G** level of theory. Among these, only one contains an intramolecular H-bond that has a small abundance at the considered temperature. Apart from this one, three other different conformations could be detected in an Ar matrix. The experimental rotamerization constants NAG2 ⇌ NAG1 and NAG3 ⇌ NAG1 could be estimated. The values of the rotamerization constants as well as the mean frequency deviation of N-acetylglycine were combined with previously obtained data of other N-acetylated amino acids and they appeared to be similar to the data for nonsubstituted amino acids. This suggests that the used methodology can be in the future applied to investigate small peptides. Analysis of H-bond frequency shifts and distance demonstrates that the intramolecular H-bonds in N-acetylated amino acids are stronger compared to those in nonsubstituted amino acids.

Small Peptide Inhibitors of Acetyl-Peptide Hydrolase Having an Uncommon Mechanism of Inhibition and a Stable Bent Conformation

Acyl peptide hydrolase (APEH) catalyzes the removal of acetyl-amino acids from the N-terminus of peptides and cytoplasmic proteins. Due to the role played in several diseases, and to the growing interest around N-terminal acetylation, studies on APEH structure, function, and inhibition are attracting an ever increasing attention. We have therefore screened a random tetrapeptide library, N-capped with selected groups, and identified a trifluoroacetylated tetrapeptide (CF(3)-lmph) which inhibits the enzyme with a K(i) of 24.0 ± 0.8 μM. The inhibitor is selective for APEH, shows an uncommon uncompetitive mechanism of inhibition, and in solution adopts a stable bent conformation. CF(3)-lmph efficiently crosses cell membranes, blocking the cytoplasmic activity of APEH; however, it triggers a mild pro-apoptotic effect as compared to other competitive and noncompetitive inhibitors. The unusual inhibition mechanism and the stable structure make the new compound a novel tool to investigate enzyme functions and a useful scaffold to develop more potent inhibitors.

A “hidden” role of amino and imino groups is unveiled during the micro-solvation study of three biomolecule groups in water

The 13C longitudinal relaxation times (T1) of three biomolecule groups of major significance to proteins and cells – protein amino acids (AAs), acetyl-amino acids (Ac-AAs) and betaines – and the 14N linewidths (Δν1/2) of Ac-AAs and betaines were measured in aqueous solutions at acidic and neutral pH, by NMR spectroscopy, to estimate the effect of the molecular weight (Mw) on 13C longitudinal relaxation times and 14N linewidths, respectively. 13C relaxation times indicate that AAs and Ac-AAs strongly interact with the same number of water molecules at acidic and neutral pH, respectively, whereas both 13C and 14N results indicate that their Mw values at acidic pH (protonated, positively charged AAs and zero-charged Ac-AAs) increase relatively to those at pH 6.0 (zwitterionic AAs and negatively charged Ac-AAs) that translates into their extra hydration with an excess of one water molecule. Both 13C and 14N relaxation times revealed that betaines retain their hydration grade in both their ionization states at two pH values, while exhibiting their hydration differences from AAs and Ac-AAs and pointing out the “controlling” role of the amino and imino groups in the extra hydration of protonated AAs and Ac-AAs, enlightening the so far unknown significant role of the N-terminus and the −NH near the C-terminus in peptide solvation. Moreover, DFT calculations of the interacting water molecules through hydrogen bonds with Ala, Ac-Ala and Ala betaine molecules in their protonated and neutral pH forms are in absolute agreement with NMR results. Finally, a fully promising method arises with a view on hydration–solvation studies of oligopeptides and other bio-organic molecules by 13C relaxation.

Dual Effects of Phloretin and Phloridzin on the Glycation Induced by Methylglyoxal in Model Systems

In the present study, the dual effects of phloretin and phloridzin on methylglyoxal (MGO)-induced glycation were investigated in three N(α)-acetyl amino acid (arginine, cysteine, and lysine) models and three N-terminal polypeptide (PP01, PP02, and PP03 containing arginine, cysteine, and lysine, respectively) models. In both N(α)-acetyl amino acids and N-terminal polypeptides models, the arginine residue was confirmed as the major target for modification induced by MGO. Meanwhile, MGO modification was significantly inhibited by the addition of phloretin or phloridzin via their MGO-trapping abilities, with phloretin being more effective. Interestingly, the cysteine residue was intact when solely incubated with MGO, whereas the consumption of N(α)-acetylcysteine and PP02 was promoted by the addition of phloretin. Additional adducts, [N(α)-acetylcysteine + 2MGO + phloretin-H(2)O] and [2N(α)-acetylcysteine + 2MGO + phloretin-2H(2)O] were formed in the model composed of N(α)-acetylcysteine, MGO, and phloretin. Another adduct, [PP02 + 2MGO + phloretin-H(2)O] was observed in the model composed of PP02, MGO, and phloretin. The generation of adducts indicates that phloretin could directly participate in the modification of the cysteine residue in the presence of MGO. When creatine kinase (model protein) was exposed to MGO, the addition of phloridzin did not show a significant effect on retaining the activity of creatine kinase impaired by MGO, whereas the addition of phloretin completely inactivated creatine kinase. Results of the mass spectrometric analysis of intact creatine kinase in different models demonstrated that phloretin could directly participate in the reaction between creatine kinase and MGO, which would lead to the inactivation of creatine kinase. Furthermore, the addition of N(α)-acetylcysteine was found to maintain the activity of creatine kinase incubated with phloretin and MGO. These results showed that phloretin and phloridzin could inhibit the modification of the arginine residue by MGO and that phloretin could directly participate in the reaction between the thiol group and MGO.

Scientific Opinion on application (EFSA‐GMO‐UK‐2007‐43) for the placing on the market of herbicide tolerant genetically modified soybean 356043 for food and feed uses, import and processing under Regulation (EC) No 1829/2003 from Pioneer

This scientific opinion is an evaluation of a risk assessment for the genetically modified herbicide tolerant soybean 356043 for food and feed uses, import and processing. Soybean 356043 contains a single copy of intact gat4601 and Glycine max-hra cassettes at a single insertion locus. The results of the bioinformatic analyses of the insert and the flanking regions, and the levels of newly expressed proteins did not raise a safety concern. The comparative analysis of phenotypic and agronomic characteristics indicated that soybean 356043 is not different from its conventional counterpart. In the composition, differences were identified between 356043 soybean and its conventional counterpart in the newly expressed proteins Glycine max-HRA and GAT4601, and the levels of the fatty acids heptadecanoic, heptadecenoic and heptadecadienoic acid and the acetylated amino acids N-acetylaspartate (NAA) and N-acetylglutamate (NAG). The safety assessment of the newly expressed proteins Glycine max-HRA and GAT4601 identified no concerns regarding potential toxicity and allergenicity. Heptadecanoic, heptadecenoic and heptadecadienoic acid are present in the diet and the intake of small amounts of these fatty acids via food or feed is not expected to produce adverse effects. NAA and NAG are normal constituents in the mammalian metabolism and the estimated increases in their intake are considered low when related to the normal intake of L-aspartic acid and L-glutamic acid. Further toxicological, allergenicity and nutritional analysis provided no indications of adverse effects. There are no indications of an increased likelihood of establishment and spread of feral soybean plants, except in the presence of the glyphosate and ALS-inhibiting herbicides neither a risk caused by a possible transfer of the recombinant gene from soybean 356043 to environmental micro-organisms. The EFSA GMO Panel considers that the information available for soybean 356043 addresses the scientific comments raised by the Member States and states that the soybean 356043, as described in this application, is as safe as its conventional counterpart with respect to potential effects on human and animal health and the environment in the context of its intended uses.