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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.39, #5)

The impact of histone post-translational modifications on developmental gene regulation by Mark N. Cruickshank; Paul Besant; Daniela Ulgiati (pp. 1087-1105).

Eukaryotic genomic DNA is orderly compacted to fit into the nucleus and to inhibit accessibility of specific sequences. DNA is manipulated in many different ways by bound RNA and proteins within the composite material known as chromatin. All of the biological processes that require access to genomic DNA (such as replication, recombination and transcription) therefore are dependent on the precise characteristics of chromatin in eukaryotes. This distinction underlies a fundamental property of eukaryotic versus prokaryotic gene regulation such that chromatin structure must be regulated to precisely repress or relieve repression of particular regions of the genome in an appropriate spatio-temporal manner. As well as playing a key role in structuring genomic DNA, histones are subject to site-specific modifications that can influence the organization of chromatin structure. This review examines the molecular processes regulating site-specific histone acetylation, methylation and phosphorylation with an emphasis on how these processes underpin differentiation-regulated transcription.

Keywords: Cell differentiation; Histones; Chromatin; Transcription factors

Lactic acid bacterial cell factories for gamma-aminobutyric acid by Haixing Li; Yusheng Cao (pp. 1107-1116).

Gamma-aminobutyric acid is a non-protein amino acid that is widely present in organisms. Several important physiological functions of gamma-aminobutyric acid have been characterized, such as neurotransmission, induction of hypotension, diuretic effects, and tranquilizer effects. Many microorganisms can produce gamma-aminobutyric acid including bacteria, fungi and yeasts. Among them, gamma-aminobutyric acid-producing lactic acid bacteria have been a focus of research in recent years, because lactic acid bacteria possess special physiological activities and are generally regarded as safe. They have been extensively used in food industry. The production of lactic acid bacterial gamma-aminobutyric acid is safe and eco-friendly, and this provides the possibility of production of new naturally fermented health-oriented products enriched in gamma-aminobutyric acid. The gamma-aminobutyric acid-producing species of lactic acid bacteria and their isolation sources, the methods for screening of the strains and increasing their production, the enzymatic properties of glutamate decarboxylases and the relative fundamental research are reviewed in this article. And the potential applications of gamma-aminobutyric acid-producing lactic acid bacteria were also referred to.

Keywords: Gamma-aminobutyric acid; Lactic acid bacteria; Glutamate decarboxylase

Towards a better understanding of nuclear processes based on proteomics by Ry Y. Tweedie-Cullen; Isabelle M. Mansuy (pp. 1117-1130).

The complex structural and functional organisation of the brain warrants the application of high-throughput approaches to study its functional alterations in physiological and pathological conditions. Such approaches have greatly benefited from advances in proteomics and genomics, and from their combination with computational modelling. They have been particularly instrumental for the analysis of processes such as the post-translational modification (PTM) of proteins, a critical biological process in the nervous system that remains not well studied. Protein PTMs are dynamic covalent marks that can be induced by activity and allow the maintenance of a trace of this activity. In the nucleus, they can modulate histone proteins and the components of the transcriptional machinery, and thereby contribute to regulating gene expression. PTMs do however need to be tightly controlled for proper chromatin functions. This review provides a synopsis of methods available to study PTMs and protein expression based on high-throughput mass spectrometry (MS), and covers basic concepts of traditional ‘shot-gun’-based MS. It describes classical and emerging proteomic approaches such as multiple reaction monitoring and electron transfer dissociation, and their application to the analyses of nuclear processes in the brain.

Keywords: Neuroproteomics; Nucleus; Neuroscience; Histones; PTMs; Signalling; Transcription; Quantitation; Mass spectrometry

Reduction of protein radicals by GSH and ascorbate: potential biological significance by Janusz M. Gebicki; Thomas Nauser; Anastasia Domazou; Daniel Steinmann; Patricia L. Bounds; Willem H. Koppenol (pp. 1131-1137).

The oxidation of proteins and other macromolecules by radical species under conditions of oxidative stress can be modulated by antioxidant compounds. Decreased levels of the antioxidants glutathione and ascorbate have been documented in oxidative stress-related diseases. A radical generated on the surface of a protein can: (1) be immediately and fully repaired by direct reaction with an antioxidant; (2) react with dioxygen to form the corresponding peroxyl radical; or (3) undergo intramolecular long range electron transfer to relocate the free electron to another amino acid residue. In pulse radiolysis studies, in vitro production of the initial radical on a protein is conveniently made at a tryptophan residue, and electron transfer often leads ultimately to residence of the unpaired electron on a tyrosine residue. We review here the kinetics data for reactions of the antioxidants glutathione, selenocysteine, and ascorbate with tryptophanyl and tyrosyl radicals as free amino acids in model compounds and proteins. Glutathione repairs a tryptophanyl radical in lysozyme with a rate constant of (1.05 ± 0.05) × 10⁵ M^–1s^–1, while ascorbate repairs tryptophanyl and tyrosyl radicals ca. 3 orders of magnitude faster. The in vitro reaction of glutathione with these radicals is too slow to prevent formation of peroxyl radicals, which become reduced by glutathione to hydroperoxides; the resulting glutathione thiyl radical is capable of further radical generation by hydrogen abstraction. Although physiologically not significant, selenoglutathione reduces tyrosyl radicals as fast as ascorbate. The reaction of protein radicals formed on insulin, β-lactoglobulin, pepsin, chymotrypsin and bovine serum albumin with ascorbate is relatively rapid, competes with the reaction with dioxygen, and the relatively innocuous ascorbyl radical is formed. On the basis of these kinetics data, we suggest that reductive repair of protein radicals may contribute to the well-documented depletion of ascorbate in living organisms subjected to oxidative stress.

Keywords: Protein oxidation; Protein repair; Ascorbate; Glutathione; Selenocysteine; Long range electron transfer

Evidence that the tri-cellular metabolism of N-acetylaspartate functions as the brain’s “operating system”: how NAA metabolism supports meaningful intercellular frequency-encoded communications by Morris H. Baslow (pp. 1139-1145).

N-acetylaspartate (NAA), an acetylated derivative of l-aspartate (Asp), and N-acetylaspartylglutamate (NAAG), a derivative of NAA and l-glutamate (Glu), are synthesized by neurons in brain. However, neurons cannot catabolize either of these substances, and so their metabolism requires the participation of two other cell types. Neurons release both NAA and NAAG to extra-cellular fluid (ECF) upon stimulation, where astrocytes, the target cells for NAAG, hydrolyze it releasing NAA back into ECF, and oligodendrocytes, the target cells for NAA, hydrolyze it releasing Asp to ECF for recycling to neurons. This sequence is unique as it is the only known amino acid metabolic cycle in brain that requires three cell types for its completion. The results of this cycling are two-fold. First, neuronal metabolic water is transported to ECF for its removal from brain. Second, the rate of neuronal activity is coupled with focal hyperemia, providing stimulated neurons with the energy required for transmission of meaningful frequency-encoded messages. In this paper, it is proposed that the tri-cellular metabolism of NAA functions as the “operating system” of the brain, and is essential for normal cognitive and motor activities. Evidence in support of this hypothesis is provided by the outcomes of two human inborn errors in NAA metabolism.

Keywords: Aquaporins; Brain; Molecular water pumps; Neurons; Free water; Bound water

Structural elucidation of Leuprolide and its analogues in solution: insight into their bioactive conformation by Despina K. Laimou; Maria Katsara; Minos-Timotheos I. Matsoukas; Vasso Apostolopoulos; Anastassios N. Troganis; Theodore V. Tselios (pp. 1147-1160).

Leuprolide [dLeu⁶, NHEt¹⁰]GnRH, a potent gonadotropin-releasing hormone (GnRH) agonist, is used in a wide variety of hormone-related diseases like cancer and endometriosis. In this report, the conformational behaviour of Leuprolide and its linear synthetic analogues, namely [Tyr⁵(OMe), dLeu⁶, Aze⁹, NHEt¹⁰]GnRH (1) and [Tyr⁵(OMe), dLeu⁶, NHEt¹⁰]GnRH (2) have been studied in DMSO and H₂O solutions by means of 2D nuclear magnetic resonance (NMR) experiments and detailed molecular dynamics (MD) simulations. The aim was to identify the conformational requirements of GnRH analogues for agonistic activity. This approach is of value as no crystallographic data are available for the GnRH receptor (G protein-coupled receptor, GPCR). The NOE data indicate the existence of a β-turn type I in the 2–5 segments of Leuprolide and its linear analogues in the case of using DMSO-d₆ as solvent, whereas a β-turn type II in the 3–6 segments is indicated using D₂O as solvent. The final structures fulfil the conformational requirements that are known, in the literature, to play a significant role in receptor recognition and activation. Finally, the linear analogues (1) and (2) are biologically active when tested against the human breast cancer cell line, MCF-7.

Keywords: Gonadotropin-releasing hormone (GnRH); Leuprolide; Nuclear magnetic resonance; Bioactive conformation; Molecular dynamics

Hydrogen sulfide protects against vascular remodeling from endothelial damage by Thomas P. Vacek; William Gillespie; Neetu Tyagi; Jonathan C. Vacek; Suresh C. Tyagi (pp. 1161-1169).

Remodeling by its very nature implied synthesis and degradation of extracellular matrix (ECM) proteins. Although oxidative stress, matrix metalloproteinase (MMP) and tissue inhibitor of metalloproteinase (TIMP) have been implicated in vascular remodeling, the differential role of MMPs versus TIMPs and oxidative stress in vascular remodeling was unclear. TIMP-3 induced vascular cell apoptosis, therefore, we hypothesized that during vascular injury TIMP-3, MMP-9 and -12 (elastin-degrading MMP) were increased, whereas MMP-2 (constitutive MMP) and TIMP-4 (cardioprotective TIMP) decreased. Because of the potent anti-oxidant, vasorelaxing, anti-hypertensive agent, hydrogen sulfide (H₂S) was used to mitigate the vascular remodeling due to the differential expression of MMP and TIMP. Carotid artery injury was created by inserting a PE-10 catheter and rotating several times before pulling out. The insertion hole was sealed. Mice were grouped: wild type (WT), wild-type damaged artery (WTD), WT + NaHS (sodium hydrogen sulfide, precursor of H₂S) treatment (30 µmol/L in drinking water/6 weeks) and WTD + NaHS treatment. Carotid arteries were analyzed for oxidative stress and remodeling, by measuring super oxide dismutase-1 (SOD1), p47 (NADPH oxidase subunit), nitrotyrosine, MMPs and TIMPs by in situ immunolabeling and by Western blot analyses. The results suggested robust increase in p47, nitrotyrosine, MMP-9, MMP-12, TIMP-3 and decrease in SOD1 and MMP-2 levels in the injured arteries. The treatment with H₂S ameliorated these effects. We concluded that p47, TIMP-3, MMP-9 and -12 were increased where as SOD-1, MMP-2 and TIMP-4 were decreased in the injured arteries. The treatment with H₂S mitigated the vascular remodeling by normalizing the levels of redox stress, MMPs and TIMPs.

Keywords: SOD; P47; NaHS; Nitrotyrosine; MMP-12; ROS; RNS; RTS

Asymmetric synthesis of enantiomerically and diastereoisomerically enriched 4-[F or Br]-substituted glutamic acids by Yuri N. Belokon; Victor I. Maleev; Tatiana F. Savel’eva; Margarita A. Moskalenko; Dmitri A. Pripadchev; Victor N. Khrustalev; Ashot S. Saghiyan (pp. 1171-1176).

A novel simple synthetic protocol for the preparation of both (2S,4R)- and (2S,4S)-FGlu, applying Michael addition of methyl α-fluoroacrylate to a Ni^II complex of glycine Schiff base with BPB, was elaborated. In addition, same reaction of mentioned complex with ethyl α-bromoacrylate leads to the Ni^II complex of the Schiff base of BPB with (2S,4R)-4-bromo-glutamic acid monoester, that can be transformed into the corresponding complexes of 1-aminocyclopropane-1,2-dicarboxylic acid. The decomposition of the diastereoisomerically pure complexes leads to corresponding enantiomerically enriched (ee > 98%) amino acids.

Keywords: Asymmetric synthesis; Chiral Ni^II Schiff base complexes; (2S)-4-[¹⁹F or ¹⁸F]fluoroglutamic acid; (1S,2R)-1-aminocyclopropane-1,2-dicarboxylic acid; (1S,2S)-1-aminocyclopropane-1,2-dicarboxylic acid; (2S,4R)-4-bromoglutamic acid

Enzymatic synthesis of theanine from glutamic acid γ-methyl ester and ethylamine by immobilized Escherichia coli cells with γ-glutamyltranspeptidase activity by Fei Zhang; Qing-Zhong Zheng; Qing-Cai Jiao; Jun-Zhong Liu; Gen-Hai Zhao (pp. 1177-1182).

Theanine (γ-glutamylethylamide) is the main amino acid component in green tea. The demand for theanine in the food and pharmaceutical industries continues to increase because of its special flavour and multiple physiological effects. In this research, an improved method for enzymatic theanine synthesis is reported. An economical substrate, glutamic acid γ-methyl ester, was used in the synthesis catalyzed by immobilized Escherichia coli cells with γ-glutamyltranspeptidase (GGT) activity. The results show that GGT activity with glutamic acid γ-methyl ester as substrate was about 1.2-folds higher than that with glutamine as substrate. Reaction conditions were optimized by using 300 mmol/l glutamic acid γ-methyl ester, 3,000 mmol/l ethylamine, and 0.1 g/ml of immobilized GGT cells at pH 10 and 50°C. Under these conditions, the immobilized cells were continuously used ten times, yielding an average glutamic acid γ-methyl ester to theanine conversion rate of 69.3%. Bead activity did not change significantly the first six times they were used, and the average conversion rate during the first six instances was 87.2%. The immobilized cells exhibited favourable operational stability.

Keywords: Theanine; Enzymatic synthesis; Glutamic acid γ-methyl ester; γ-Glutamyltranspeptidase

Gluten T cell epitope targeting by TG3 and TG6; implications for dermatitis herpetiformis and gluten ataxia by Jorunn Stamnaes; Siri Dorum; Burkhard Fleckenstein; Daniel Aeschlimann; Ludvig M. Sollid (pp. 1183-1191).

Transglutaminase 2 (TG2) is well characterized as the main autoantigen of celiac disease. The ability of TG2 to deamidate and crosslink gluten peptides is essential for the gluten-dependent production of TG2 specific autoantibodies. In patients with primarily extraintestinal manifestation of gluten sensitivity the repertoire of autoantibodies may be different. In dermatitis herpetiformis (DH), TG3 appears to be the target autoantigen whereas in gluten ataxia (GA) autoantibodies reactive with TG6 are present. A functional role for TG3 and TG6 in these diseases has yet to be described. It is also not known whether these enzymes can use gluten peptides implicated in the pathology as substrates. We here report that similar to TG2, TG3 and TG6 can specifically deamidate gluten T cell epitopes. However, the fine specificities of the enzymes were found to differ. TG2 can form covalent complexes with gluten by iso-peptide and thioester bonds. We found that both TG3 and TG6 were able to complex with gluten peptides through thioester linkage although less efficiently than TG2, whereas TG6 but not TG3 was able to form iso-peptide linked complexes. Our findings lend credence to the notion that TG3 and TG6 are involved in the gluten-induced autoimmune responses of DH and GA.

Keywords: Gluten; Autoantibodies; TG3; TG6; Dermatitis herpetiformis; Gluten ataxia

Apelin suppresses apoptosis of human vascular smooth muscle cells via APJ/PI3-K/Akt signaling pathways by Rong-Rong Cui; Ding-An Mao; Lu Yi; Cheng Wang; Xing-Xing Zhang; Hui Xie; Xian-Ping Wu; Xiao-Bo Liao; Hua Zhou; Ji-Cai Meng; Ling-Qing Yuan; Er-Yuan Liao (pp. 1193-1200).

Apoptosis of vascular smooth muscle cells (VSMCs) plays an important role in regulating vascular remodeling during cardiovascular diseases. Apelin is the endogenous ligand for the G-protein-coupled receptor APJ and plays an important role in the cardiovascular system. However, the mechanisms of apelin on apoptosis of VSMCs have not been elucidated. Using a culture of human VSMCs as a model for the study of apoptosis, the relationship between apelin and apoptosis of human VSMCs and the signal pathway involved were investigated. Using western blotting, we confirmed that VSMCs could express APJ. To evaluate the possible role of apelin in VSMC apoptosis, we assessed its effect on apoptosis of human VSMCs. The results showed that apelin inhibited human VSMCs apoptosis induced by serum deprivation. Suppression of APJ with small-interfering RNA (siRNA) abolished the anti-apoptotic activity of apelin. Apelin increased Bcl-2 protein expression, but decreased Bax protein expression. An increase in activation of extracellular signal-regulated protein kinase (ERK) and Akt (a downstream effector of phosphatidylinositol 3-kinase) was shown after apelin stimulation. Suppression of APJ with siRNA abolished the apelin-induced activation of ERK and Akt. LY294002 (a PI3-K inhibitor) blocked apelin-induced activation of Akt and abolished the apelin-induced antiapoptotic activity. Our study suggests that apelin suppresses serum deprivation-induced apoptosis of human VSMCs, and that the anti-apoptotic action is mediated through the APJ/PI3-K/Akt signaling pathways.

Keywords: Apelin; Vascular smooth muscle cells; Apoptosis; Extracellular signal-regulated kinases; Phosphatidylinositol 3-kinase; Akt

Utilization of amino acids by bacteria from the pig small intestine by Zhao-Lai Dai; Jing Zhang; Guoyao Wu; Wei-Yun Zhu (pp. 1201-1215).

This study determined the utilization of amino acids (AA) by bacteria from the lumen of the pig small intestine. Digesta samples from different segments of the small intestine were inoculated into media containing 10 mmol/L each of select AA (l-lysine, l-threonine, l-arginine, l-glutamate, l-histidine, l-leucine, l-isoleucine, l-valine, l-proline, l-methionine, l-phenylalanine or l-tryptophan) and incubated for 24 h. The previous 24-h culture served as an inoculum for a subsequent 24-h subculture during each of 30 subcultures. Results of the in vitro cultivation experiment indicated that the 24-h disappearance rates for lysine, arginine, threonine, glutamate, leucine, isoleucine, valine or histidine were 50–90% in the duodenum, jejunum or ileum groups. After 30 subcultures, the 24-h disappearance rates for lysine, threonine, arginine or glutamate remained greater than 50%. The denaturing gradient gel electrophoresis analysis showed that Streptococcus sp., Mitsuokella sp., and Megasphaera elsdenii-like bacteria were predominant in subcultures for utilizing lysine, threonine, arginine and glutamate. In contrast, Klebsiella sp. was not a major user of arginine or glutamate. Furthermore, analysis of AA composition and the incorporation of AA into polypeptides indicated that protein synthesis was a major pathway for AA metabolism in all the bacteria studied. The current work identified the possible predominant bacterial species responsible for AA metabolism in the pig small intestine. The findings provide a new framework for future studies to characterize the metabolic fate of AA in intestinal microbes and define their nutritional significance for both animals and humans.

Keywords: Pig; Small intestine; Metabolism; Bacterial community

Tanshinone IIA enhances BMP-2-stimulated commitment of C2C12 cells into osteoblasts via p38 activation by Hye Joo Kim; Seong Hwan Kim (pp. 1217-1226).

In this study, we demonstrate a stimulatory effect of tanshinone IIA isolated from the root of Salvia miltiorrhiza on the commitment of bi-potential mesenchymal precursor C2C12 cells into osteoblasts in the presence of bone morphogenetic protein (BMP)-2. At low concentrations, tanshinone IIA enhanced BMP-2-stimulated induction of alkaline phosphatase (ALP), an early phase biomarker of osteoblast differentiation, and mRNA expression of BMPs. ALP induction was inhibited by the BMP antagonist noggin, suggesting that tanshinone IIA enhances the osteogenic activity of BMP signaling. Furthermore, considering the tanshinone IIA-mediated enhancement of BMP-2-stimulated Smad-Runx2 activities, tanshinone IIA could enhance the osteogenic activity of BMP-2 via acceleration of Smad-Runx2 activation. Additionally, pharmacologic inhibition studies suggest the possible involvement of p38 in the action of tanshinone IIA. The p38 inhibitor SB202190 strongly and dose-dependently inhibited tanshinone IIA-enhanced ALP induction. SB202190 also dose-dependently inhibited the tanshinone IIA-induced p38 activation and combined tanshinone IIA-BMP-2-induced Smad activation. In conclusion, tanshinone IIA enhances the commitment of C2C12 cells into osteoblasts and their differentiation through synergistic cross talk between tanshinone IIA-induced p38 activation and BMP-2-induced Smad activation. These activations could subsequently induce the activation of Runx2, which induces osteogenesis via regulation of the osteogenic factors BMP and ALP expression.

Keywords: Tanshinone IIA; Osteoblast differentiation; BMP; p38; Smad; Runx2

A new C-type lectin (FcLec5) from the Chinese white shrimp Fenneropenaeus chinensis by Wen-Teng Xu; Xian-Wei Wang; Xiao-Wen Zhang; Xiao-Fan Zhao; Xiao-Qiang Yu; Jin-Xing Wang (pp. 1227-1239).

C-type lectins are one family of pattern recognition receptors (PRRs) that play important roles in innate immunity. In this work, cDNA and genomic sequences for a new C-type lectin (FcLec5) were obtained from the Chinese white shrimp Fenneropenaeus chinensis. FcLec5 cDNA contains an open reading frame of 1,008 bp and its genomic sequence is 1,137 bp with 4 exons and 3 introns. The predicted FcLec5 protein contains a signal peptide and two carbohydrate recognition domains (CRDs). The N-terminal CRD of FcLec5 has a predicted carbohydrate recognition motif of Gln-Pro-Asp (QPD), while the C-terminal CRD contains a motif of Glu-Pro-Gln (EPQ). Northern blot analysis showed that FcLec5 mRNA was specifically expressed in hepatopancreas. FcLec5 protein was expressed in hepatopancreas and secreted into hemolymph. Real-time PCR showed that FcLec5 transcript exhibited different expression profiles after immune-challenged with Vibrio anguillarum or White Spot Syndrome Virus (WSSV). Recombinant FcLec5 and its two individual CRDs could agglutinate most bacteria tested, and the agglutinating activity was Ca²⁺-dependent. Besides, the agglutinating activity to gram-negative bacteria is higher than that to gram-positive bacteria. Direct binding assay showed that recombinant FcLec5 could bind to all microorganisms tested (five gram-positive and four gram-negative bacteria, as well as yeast) in a Ca²⁺-independent manner. Recombinant FcLec5 also directly bound to bacterial peptidoglycan, lipopolysaccharide and lipoteichoic acids. These results suggest that FcLec5 may act as a PRR for bacteria via binding to bacterial cell wall polysaccharides in Chinese white shrimp.

Keywords: C-type lectin; Innate immunity; Expression profile; Binding spectrum; Sugar specificity; Fenneropenaeus chinensis

Influence of assignment on the prediction of transmembrane helices in protein structures by Jean Pylouster; Aurélie Bornot; Catherine Etchebest; Alexandre G. de Brevern (pp. 1241-1254).

α-Helical transmembrane proteins (TMP_α) are composed of a series of helices embedded in the lipid bilayer. Due to technical difficulties, few 3D structures are available. Therefore, the design of structural models of TMP_α is of major interest. We study the secondary structures of TMP_α by analyzing the influence of secondary structures assignment methods (SSAMs). For this purpose, a published and updated benchmark databank of TMP_α is used and several SSAMs (9) are evaluated. The analysis of the results points to significant differences in SSA depending on the methods used. Pairwise comparisons between SSAMs led to more than 10% of disagreement. Helical regions corresponding to transmembrane zones are often correctly characterized. The study of the sequence–structure relationship shows very limited differences with regard to the structural disagreement. Secondary structure prediction based on Bayes’ rule and using only a single sequence give correct prediction rates ranging from 78 to 81%. A structural alphabet approach gives a slightly better prediction, i.e., only 2% less than the best equivalent approach, whereas the prediction rate with a very different assignment bypasses 86%. This last result highlights the importance of the correct assignment choice to evaluate the prediction assessment.

Keywords: Amino acid; Secondary structure; Secondary structure assignment method; DSSP; Transmembrane protein; Molecular modeling; Structural alphabet

Synthesis and characterization of novel, optically active polyamides derived from S-valine natural amino acid and bulky anthracenic side chain by Shadpour Mallakpour; Fatemeh Mirkarimi (pp. 1255-1263).

This is the first description of the application of molten tetrabutylammonium bromide (TBAB) in the presence of triphenyl phosphite (TPP) for the synthesis of novel polyamides (PAs). Monomer diacid, 5-[(9,10-dihydro-9,10-ethanoanthracene-11,12-dicarboximido)-3-methylbutanoylamino]isophthalic acid (4), having anthracenic and amino acid S-valine pendant group, was synthesized in four steps. Several novel, optically active PAs were prepared by the condensation of synthesized diacid monomer 4 with various aromatic diamines using two different techniques: a mixture of N-methyl-2-pyrrolidone (NMP)/TPP/pyridine/calcium chloride (method I) and combination of TPP with TBAB (method II). The main goal of the present paper was to prepare novel PAs in a green media by removal of toxic reagents. Therefore, TBAB/TPP was used as a novel, easy, safe and eco-friendly method for the preparation of aromatic PAs. This method is compared with the polymerization reaction under conventional solvent and in the case of TBAB as a new method, higher yields, inherent viscosities and thermally stable of PAs are gained. The resulting polymers showed good solubility in polar aprotic solvents such as dimethyl sulfoxide, NMP, N,N-dimethylacetamide and N,N-dimethylformamide. These polymers are characterized with respect to chemical structure and purity by means of specific rotation experiments, FT-IR, ¹H NMR spectroscopy techniques and elemental analysis. The obtained PAs exhibit good thermal stability up to 335°C for 10% weight loss in nitrogen atmosphere and glass transition temperatures fell in the rang of 177–185°C.

Keywords: Green chemistry; Tetrabutylammonium bromide; Optically active polyamides; Phosphorylation polycondensation; S-Valine

Synthesis of α-carboxyphosphinopeptides derived from norleucine by Jan Pícha; Miloš Buděšínský; Pavel Fiedler; Miloslav Šanda; Jiří Jiráček (pp. 1265-1280).

In the present study, we describe in detail the synthesis of a relatively rare class of phosphorus compounds, α-carboxyphosphinopeptides. We prepared several norleucine-derived α-carboxyphosphinic pseudopeptides of the general formula Nle-Ψ[PO(OH)]-Gly. These compounds could have important applications as transition state-mimicking inhibitors for methionine or leucine aminopeptidases or other enzymes. For the preparation of the key α-carboxyphosphinate protected precursors, we investigated, compared and improved two different synthetic methods described in literature: the Arbuzov reaction of a silylated N-protected phosphinic acid with a bromoacetate ester and the nucleophilic addition of a mixed O-methyl S-phenyl N-protected phosphonic acid or a methyl N-protected phosphonochloridate with tert-butyl lithioacetate. We also prepared two N-Fmoc protected synthons, Fmoc-Nle-Ψ[PO(OH)]-Gly-COOH and Fmoc-Nle-Ψ[PO(OAd)]-Gly-COOH, and demonstrated that these precursors are suitable building blocks for the solid-phase synthesis of α-carboxyphosphinopeptides.

Keywords: Phosphinopeptides; Pseudopetides; Phosphinate; Phosphinic pseudopeptides; Solid-phase synthesis; Norleucine

Distribution of methionine between cells and incubation medium in suspension of rat hepatocytes by Tatyana K. Korendyaseva; Michael V. Martinov; Alexander M. Dudchenko; Victor M. Vitvitsky (pp. 1281-1289).

Methionine is an essential amino acid involved in many significant intracellular processes. Aberrations in methionine metabolism are associated with a number of complex pathologies. Liver plays a key role in regulation of blood methionine level. Investigation of methionine distribution between hepatocytes and medium is crucial for understanding the mechanisms of this regulation. For the first time, we analyzed the distribution of methionine between hepatocytes and incubation medium using direct measurements of methionine concentrations. Our results revealed a fast and reversible transport of methionine through the cell membrane that provides almost uniform distribution of methionine between hepatocytes and incubation medium. The steady-state ratio between intracellular and extracellular methionine concentrations was established within a few minutes. This ratio was found to be 1.06 ± 0.38, 0.89 ± 0.26, 0.67 ± 0.16 and 0.82 ± 0.06 at methionine concentrations in the medium of 64 ± 19, 152 ± 39, 413 ± 55, and 1,204 ± 104 μmol/L, respectively. The fast and uniform distribution of methionine between hepatocytes and extracellular compartments provides a possibility for effective regulation of blood methionine levels due to methionine metabolism in hepatocytes.

Keywords: Methionine transport; Methionine distribution; Hepatocytes; Rat; Liver

Mitomycin C modulates DNA-double strand break repair genes in cervical carcinoma cells by Yun Hee Kang; Kyung-Ae Lee; Jung-Hee Kim; Sung-Goo Park; Do-Young Yoon (pp. 1291-1298).

In a previous study, we elucidated the apoptotic mechanism mediated via Fas/FasL-dependent pathway in mitomycin C-treated cervical carcinoma cells. In this study, 2-D and MALDI-TOF analyses were performed in order to search mitomycin C-induced modulators in cervical carcinoma cells. Some protein spots down- or up-regulated by mitomycin C were separately selected from the 2-D gels. Twenty protein spots were identified from the 2-D gels. Among the 20 spots, 11 spots were down-regulated, whereas 9 spots were up-regulated in SiHa/pRSV-luc cells by mitomycin C. Three spots have not been identified in the database. Ku70-binding protein (KUB3), MHC class I antigen, MHC class I chain-related protein A or multi-PDZ domain protein 1, MAGUK P55 subfamily member 3 or lamda/iota protein kinase C-interacting protein, and GL014 or Sad1/unc-84 protein-like 1 were suppressed by mitomycin C treatment. Heat shock 60 kDa protein 1 (chaperonin), similar to heat shock protein 90 kDa protein alpha or ninein centrosomal protein isoform C, NADP-dependent malic enzyme, mitochondrial precursor, GRB10 adaptor protein, glycogenin-interacting protein 1, cystathionine gamma-lyase, G₂/mitotic-specific cyclin B2 or heat shock 90 kDa protein 1 alpha, peptidyl-prolyl cis–trans isomerase B, and PARP-2 (fragment) were induced by mitomycin C. KUB3, Brca1, and E6 gene expressions were down-regulated by mitomycin C in HPV-positive cervical cancer cells, SiHa/pRSV-luc and SiHa. In these studies, we suggest that MMC down-regulated the expression levels of the upstream molecules of DNA-double strand break repair system, non-homologous end joining or homologous recombination, resulting in the suppression of cervical cancer cell growth.

Keywords: Mitomycin C; DSB repair genes; Ku70-binding protein; Cervical carcinoma cells

2-Alkyl-2-carboxyazetidines as γ-turn inducers: incorporation into neurotrophin fragments by José Luis Baeza; M. Ángeles Bonache; M. Teresa García-López; Rosario González-Muñiz; Mercedes Martín-Martínez (pp. 1299-1307).

Conveniently substituted 2-alkyl-2-carboxyazetidine amino acids have been incorporated into NGF and NT3 tetrapeptide sequences to investigate their utility as reverse turn inducers (γ- vs. β-turns). Despite the presence of an Asp residue at i position, highly preferred in β-turns, molecular modeling and NMR studies indicated that the azetidine-containing peptides mainly stabilized γ-turn conformations.

Keywords: 2-Alkylazetidines; Reverse γ-turn inducers; Conformationally restricted amino acids; Peptides

Effect of metal ions (Li⁺, Na⁺, K⁺, Mg²⁺, Ca²⁺, Ni²⁺, Cu²⁺ and Zn²⁺) and water coordination on the structure and properties of l-histidine and zwitterionic l-histidine by Milan Remko; Daniel Fitz; Bernd Michael Rode (pp. 1309-1319).

Interactions between metal ions and amino acids are common both in solution and in the gas phase. The effect of metal ions and water on the structure of l-histidine is examined. The effect of metal ions (Li⁺, Na⁺, K⁺, Mg²⁺, Ca²⁺, Ni²⁺, Cu²⁺ and Zn²⁺) and water on structures of His·M(H₂O)m, m = 0.1 complexes have been determined theoretically employing density functional theories using extended basis sets. Of the five stable complexes investigated the relative stability of the gas-phase complexes computed with DFT methods (with one exception of K⁺ systems) suggest metallic complexes of the neutral l-histidine to be the most stable species. The calculations of monohydrated systems show that even one water molecule has a profound effect on the relative stability of individual complexes. Proton dissociation enthalpies and Gibbs energies of l-histidine in the presence of the metal cations Li⁺, Na⁺, K⁺, Mg²⁺, Ca²⁺, Ni²⁺, Cu²⁺ and Zn²⁺ were also computed. Its gas-phase acidity considerably increases upon chelation. Of the Lewis acids investigated, the strongest affinity to l-histidine is exhibited by the Cu²⁺ cation. The computed Gibbs energies ΔG are negative, span a rather broad energy interval (from −130 to −1,300 kJ/mol), and upon hydration are appreciably lowered.

Keywords: Histidine; Li⁺, Na⁺, K⁺, Mg²⁺, Ca²⁺, Ni²⁺, Cu²⁺ and Zn²⁺ complexes; DFT; Interaction enthalpy and Gibbs energy; Hydration

Purification, characterization, structural analysis and protein chemistry of a buffalo heart galectin-1 by Ghulam Md Ashraf; Sabika Rizvi; Shoa Naqvi; Nida Suhail; Nayeem Bilal; Shirin Hasan; Mohammad Tabish; Naheed Banu (pp. 1321-1332).

A soluble β-galactoside-binding lectin was purified by gel filtration chromatography from Bubalus bubalis heart. Its metal-independent nature, molecular weight of 14.5 kDa, preferential affinity for β-d-lactose, and 87–92% identity with carbohydrate recognition domain of previously reported galectin-1 confirmed its inclusion in galectin-1 subfamily. Stokes radii determination using gel filtration under reducing and non-reducing conditions revealed its homo-dimeric nature, further confirming its Gal-1 nomenclature. The purified lectin was found to be the most stable mammalian heart galectin purified till date, suggesting its preferential use in various recognition studies. Treatment of the purified lectin with oxidizing agent, thiol blocking reagents, denaturants, and detergents resulted in significant changes in UV–VIS, fluorescence, CD and FTIR spectra, which strongly emphasized the important aspect of regular secondary structure of galectins for the maintenance of their active conformation. Reduction of the activity of the purified lectin after oxidation by H₂O₂, with remarkable fluorescence quenching, may suggest potential role for galectin-1 in free radical-induced, oxidative stress-mediated cardiovascular disorders. The predictions of bioinformatics studies were found to be in accordance with the results obtained in wet lab.

Keywords: Buffalo heart galectin-1 (B_fHG-1); Bubalus bubalis ; Purification; Oxidation; Sequencing; 3D-JIGSAW; Cardiovascular disorders

Evaluation of different generic in silico methods for predicting HLA class I binding peptide vaccine candidates using a reverse approach by Uthaman Gowthaman; Sathi Babu Chodisetti; Pankaj Parihar; Javed N. Agrewala (pp. 1333-1342).

Since CD8⁺ T cell response is crucial to combat intracellular infections and cancer, identification of class I HLA binding peptides is of immense clinical value. The experimental identification of such peptides is protracted and laborious. Exploiting in silico tools to discover such peptides is an attractive alternative. However, this approach needs a thorough assessment before its elaborate application. We have adopted a reverse approach to evaluate the reliability of eight different servers (inclusive of 55 predictors) by exploiting experimentally proven data. A comprehensive data set of more than 960 peptides was employed to test the efficacy of the programs. We have validated commonly used strategies to predict peptides that bind to seven most prevalent HLA class I alleles. We conclude that four of the eight servers are more adept in predictions. Although the overall predictions for class I MHC binders were superior to class II MHC binders, individual predictors for different alleles belonging to the same program were highly variable in their efficiencies. We have also addressed whether a consensus approach can yield better prediction efficiency. We observed that combining the results from different in silico programs could not increase the efficiency significantly.

Keywords: HLA; In silico methods; Promiscuous peptides

Design and recombinant expression of insulin-like peptide 5 precursors and the preparation of mature human INSL5 by Xiao Luo; Ross A. D. Bathgate; Wei-Jie Zhang; Ya-Li Liu; Xiao-Xia Shao; John D. Wade; Zhan-Yun Guo (pp. 1343-1352).

Insulin-like peptide 5 (INSL5) is a recently identified insulin superfamily member. Although it binds to and activates the G-protein coupled receptor, RXFP4, its precise biological function remains unknown. To help determine its function, significant quantities of INSL5 are required. In the present work, three single-chain INSL5 precursors were designed, two of which were successfully expressed in E. coli cells. The expressed precursors were solubilized from inclusion bodies, purified almost to homogeneity by immobilized metal-ion affinity chromatography, and then refolded in vitro. One precursor could be converted to two-chain human INSL5 bearing an extended N-terminus of the A-chain (designated long-INSL5) by sequential Lys-C endoproteinase and carboxypeptidase B treatment. The 6 residue A-chain N-terminal extension of long-INSL5 was subsequently removed by Aeromonas aminopeptidase to yield native INSL5 that was designated short-INSL5. Circular dichroism spectroscopic analysis and peptide mapping showed that the recombinant INSL5s adopted an insulin-like conformation and possessed the expected characteristic insulin-like disulfide linkages. Activity assay showed that both long- and short-INSL5 had full RXFP4 receptor activity compared with chemically synthesized human INSL5. This suggested that extension of the N-terminus of the A-chain of long-INSL5 did not adversely impact upon the binding to or activation of the RXFP4 receptor. However, the single-chain INSL5 precursor was inactive which indicated that a free C-terminus of the B-chain is critical for the activity of INSL5. Our present work thus provides an efficient approach for preparation of INSL5 and its analogs through recombinant expression in E. coli cells.

Keywords: Activity; INSL5; Recombinant expression; Single-chain precursor; Refolding

Prediction of catalytic residues based on an overlapping amino acid classification by Yongchao Dou; Xiaoqi Zheng; Jialiang Yang; Jun Wang (pp. 1353-1361).

Protein sequence conservation is a powerful and widely used indicator for predicting catalytic residues from enzyme sequences. In order to incorporate amino acid similarity into conservation measures, one attempt is to group amino acids into disjoint sets. In this paper, based on the overlapping amino acids classification proposed by Taylor, we define the relative entropy of Venn diagram (RVD) and RVD2. In large-scale testing, we demonstrate that RVD and RVD2 perform better than many existing conservation measures in identifying catalytic residues, especially than the commonly used relative entropy (RE) and Jensen–Shannon divergence (JSD). To further improve RVD and RVD2, two new conservation measures are obtained by combining them with the classical JSD. Experimental results suggest that these combination measures have excellent performances in identifying catalytic residues.

Keywords: Catalytic sites prediction; Sequence conservation; Stereochemical properties; Overlapping sets; Combination measure

In situ fabrication and electrochemical behavior of amino acid polyoxometalate nanoparticles-embedded microcapsules by Dawei Fan; Yongming Deng; Jingcheng Hao (pp. 1363-1367).

Amino acid polyoxometalate nanoparticles-embedded microcapsules were in situ fabricated by layer-by-layer (LbL) self-assembly method [polyoxometalate, H₃PMo₁₂O₄₀·nH₂O (PMo₁₂); amino acid, glycine (Gly)]. The morphology of the obtained microcapsules was characterized by transmission electron microscopy and scanning electron microscopy. The electrochemical behavior of the amino acid polyoxometalate nanoparticles-embedded microcapsules was studied by cyclic voltammetry. The microcapsules show the pH-dependent properties, indicating that the pH of solution plays an important role in the electrochemical behavior of heteropolyanions.

Keywords: Glycine; Polyoxometalate; Layer-by-layer; Self-assembly; Microcapsule

Synthesis and structural studies of new analogues of PTH(1–11) containing C^α-tetra-substituted amino acids in position 8 by Andrea Caporale; Mattia Sturlese; Elisabetta Schievano; Stefano Mammi; Evaristo Peggion (pp. 1369-1379).

The N-terminal 1–34 fragment of parathyroid hormone (PTH) is fully active in vitro and in vivo and it can reproduce all biological responses characteristic of the native intact PTH. Recently, analogues of PTH(1–11) fragments with helicity-enhancing substitutions have been demonstrated to yield potent analogues of PTH(1–34). The work describes the synthesis, biological activity and structure of analogues of the best modified PTH sequence H-Aib-Val-Aib-Glu-Ile-Gln-Leu-Nle-His-Gln-Har-NH₂ (I). In particular, the effect of the Ala/Aib substitution at positions 1 and 3 as well as of the replacement of Nle in position 8 with d-Nle, l-(αMe)-Nle and d-(αMe)-Nle was studied. The resulting peptides were characterized structurally by CD spectroscopy, solution NMR and MD, and in vitro for activity with respect to the cognate receptor, parathyroid hormone receptor.

Keywords: SPPS; CD; NMR; MD; Calpha-tetra-substituted Amino Acids; PTH

Synthesis of bioorthogonal and crosslinking amino acids for use in peptide synthesis by G. S. M. Sundaram; Ian R. Morgan; Eric M. Tippmann (pp. 1381-1384).

The ability to incorporate non-canonical amino acids into proteins by genetic or chemical methods allows one to introduce novel chemical properties into a protein at a defined residue. Such a residue may then be modified using common organic transformations. In this way, the structure or function of the peptide may be altered without perturbing any of the other neighbouring amino acids in the peptide chain. Here, we describe the syntheses and potential applications of multiple para-substituted phenylalanine derivatives comprising an isothiocyanate, α-diazoketone, or nitrone functionality. In all, three novel amino acids were synthesized in good overall yields. These non-canonical amino acids permit the further development of in vitro and in vivo chemoselective and regioselective bioconjugate reactions not possible with other reagents.

Keywords: Non-canonical amino acids; Expanded genetic code; Bioconjugate chemistry

An approach for classification of highly imbalanced data using weighting and undersampling by Ashish Anand; Ganesan Pugalenthi; Gary B. Fogel; P. N. Suganthan (pp. 1385-1391).

Real-world datasets commonly have issues with data imbalance. There are several approaches such as weighting, sub-sampling, and data modeling for handling these data. Learning in the presence of data imbalances presents a great challenge to machine learning. Techniques such as support-vector machines have excellent performance for balanced data, but may fail when applied to imbalanced datasets. In this paper, we propose a new undersampling technique for selecting instances from the majority class. The performance of this approach was evaluated in the context of several real biological imbalanced data. The ratios of negative to positive samples vary from ~9:1 to ~100:1. Useful classifiers have high sensitivity and specificity. Our results demonstrate that the proposed selection technique improves the sensitivity compared to weighted support-vector machine and available results in the literature for the same datasets.

Keywords: Imbalanced datasets; SVM; Undersampling technique

Influence of lβ-, dα- and dβ-Asp isomers of the Asp-76 residue on the properties of αA-crystallin 70–88 peptide by Noriko Fujii; Norihiko Fujii; Masashi Kida; Tadatoshi Kinouchi (pp. 1393-1399).

Proteins have been considered to consist exclusively of l-amino acids in living tissues. However, our previous studies showed that two specific aspartyl (Asp) residues in αA- and αB-crystallins from human eye lenses invert to the d-isomers to a high degree during aging. The reaction is also accompanied by isomerization into a form containing β-Asp (isoaspartate) residues. The appearance of d- and β-Asp in a protein potentially induces large changes to the higher order structure of the protein as well as to its function. However, it remains unclear whether the formation of the Asp isomer is the direct trigger of the change to the higher order structure and function. In this study, in order to clarify the effect of the inversion to d-isomers in a protein, we synthesized peptides corresponding to the 70–88 (KFVIFLDVKHFSPEDLTVK) fragment of human αA-crystallin and its corresponding diastereoisomers in which lα-Asp was replaced with lβ-Asp, dα-Asp, and dβ-Asp at position 76 and compared their biochemical properties with that of normal peptide. The peptides containing abnormal isomers (lβ-Asp, dα-Asp, and dβ-Asp residues, respectively) were more hydrophilic than the normal peptide (containing lα-Asp), lost β-sheet structure and changed to random structures. The normal peptide promoted the aggregation of insulin while the other three isomers suppressed the aggregation of insulin. This is the first evidence that a single substitution of an Asp isomer in a peptide induces a large change to the properties of the peptide.

Keywords: αA-Crystallin; d-Aspartic acid; Isomerization; Racemization

Modulation of blackspot seabream (Pagellus bogaraveo) intermediary metabolic pathways by dispensable amino acids by A. Cláudia Figueiredo-Silva; Geneviève Corraze; Sadasivam Kaushik; José B. Peleteiro; Luísa M. P. Valente (pp. 1401-1416).

The objective of the present work is to investigate the main metabolic pathways by which dispensable amino acids (DAA) are diverted towards lipid formation in blackspot seabream. For that purpose, a control diet was formulated to contain 45% of crude protein (7.2 g N/100 g dry matter) mainly supplied by fish meal (45P). In two other diets, 22.2% of the dietary nitrogen (1.6 g N/100 g dry matter) was replaced by an equivalent amount of nitrogen provided by two different mixtures of DAA: alanine and serine (diet AS) or aspartic and glutamic acid (diet AG). A fourth diet (diet 35P) only containing 35% of crude protein (5.6 g N/100 g dry matter) was included in order to analyze the possible additive effects of DAA. Compared to fish fed diet 35P, blackspot seabream appear to make a more efficient use of the nitrogen provided by alanine and serine than that provided by aspartic and glutamic acids in terms of growth. Contrary to fish fed AG, fish fed AS attained similar specific FAS activities as 45P fed fish, suggesting a further role of alanine and serine on this lipogenic pathway. Dietary nitrogen reduction (45P vs. 35P) or its replacement by a mixture of aspartic and glutamic acids (diet AG) were shown to up-regulate phosphoenolpyruvate carboxykinase (PEPCK) but without, however, any effect on plasma glucose levels. Dietary nitrogen level and nature seems to exert a complex regulation on energetic pathways through the gluconeogenesis/tricarboxylic acids cycle interaction.

Keywords: Blackspot seabream; Dietary nitrogen replacement; DAA; Intermediary metabolism

Adaptative response of nitrogen metabolism in early endotoxemia: role of ornithine aminotransferase by Gabrielle Ventura; C. Moinard; F. Segaud; S. Le Plenier; L. Cynober; J. P. De Bandt (pp. 1417-1426).

Arginine (Arg) and glutamine (Gln) utilization is greatly increased during catabolic stress. While the supply of both amino acids has been advocated in this situation, arginine administration is possibly associated with deleterious effects. From a metabolic point of view, these two amino acids are reciprocal precursors via ornithine aminotransferase (OAT). We hypothesized that OAT plays a key role in the interconversion between Arg and Gln. To test this hypothesis, we evaluated the influence of OAT activity in a model of septic shock induced by intraperitoneal injection of lipopolysaccharide (LPS) in wild-type (WT) and transgenic mice overexpressing OAT (OAT) in the liver, kidney and intestine, i.e. the three main organs of OAT expression. Plasma and tissue amino acid concentrations and tissue OAT expression and activity were measured. Five hours after LPS injection, WT and OAT mice showed a similar response to LPS in terms of inflammatory cytokine production and protein catabolism, suggesting that the interconversion between Arg and Gln through this pathway remains limited. Endotoxemia led to a significant decrease in plasma Orn levels and an increase in liver Orn levels. Of note, Orn levels were always lower in OAT mice. While only plasma Arg and Gln remained unaffected by LPS treatment, hepatic Gln was significantly increased without any difference between the two genotypes. In this model of early endotoxemia, arginine and glutamine maintained their metabolic homeostasis. Our results show an inhibition of OAT activity and expression in the liver following LPS treatment. These data highlight the importance of OAT in ornithine metabolism, especially in the liver, and suggest a post-transcriptional regulation of OAT by LPS in the liver.

Keywords: Arginine; Ornithine; Glutamine; Amino acid metabolism; Endotoxemia; Stress

Transglutaminase 2 silencing reduced the beta-amyloid-effects on the activation of human THP-1 cells by Monica Currò; Nadia Ferlazzo; Salvatore Condello; Daniela Caccamo; Riccardo Ientile (pp. 1427-1433).

The aberrant expression and activation of transglutaminase 2 (TG2), the ubiquitous enzyme which catalyzes calcium-dependent protein cross-linking reactions, has been reported in many inflammatory diseases. Chronic inflammation, mediated by prolonged activation of brain-resident immunocompetent cells, appears to be involved in the pathogenesis of several age-related diseases, such as Alzheimer’s disease. Given that increased TG2 expression has been observed in AD brains, this study was aimed to characterize the role of TG2 in THP-1 monocytes stimulated with amyloid-beta (Aβ). Aβ_1–42 treatment dose-dependently increased TG2 expression in THP-1 cells. In particular, a fivefold up-regulation of TG2, compared with control cells, was observed in the presence of 0.5 μM Aβ_1–42. At the same concentration, Aβ_1–42 was able to promote monocyte maturation as suggested by increased expression of the cell surface antigen CD14 as well as the adhesion-promoting factor fibronectin. The stimulation of THP-1 cells with Aβ_1–42 also led to a significant up-regulation of tumor necrosis factor α (TNF-α) and matrix metalloproteinase 9 (MMP-9). Interestingly, THP-1 cell transfection with small interfering RNA directed against TG2 was able to reduce Aβ_1–42 increased levels of all the examined markers of monocyte maturation (CD14, fibronectin), and activation (TNF-α, MMP-9). These results indicate that TG2 up-regulation is required for the functional THP-1 monocyte activation induced by Aβ_1–42. This work suggests that TG2 inhibition may represent a therapeutic target to ameliorate the inflammation and progression in Alzheimer’s disease.

Keywords: Transglutaminase 2; Amyloid-beta; THP-1 monocyte activation; Inflammation; Transglutaminase 2 silencing

Comparative proteomics analysis of differentially expressed proteins in soybean cell wall during flooding stress by Setsuko Komatsu; Yukito Kobayashi; Keito Nishizawa; Yohei Nanjo; Kiyoshi Furukawa (pp. 1435-1449).

Flooding is a major problem for soybean crop as it reduces the growth and grain yield. To investigate the function of the soybean cell wall in the response to flooding stress, cell wall proteins were analyzed. Cell wall proteins from roots and hypocotyls of soybeans, which were germinated for 2 days and subjected to 2 days of flooding, were purified, separated by two-dimensional polyacrylamide gel electrophoresis and stained with Coomassie brilliant blue. Sixteen out of 204 cell wall proteins showed responses to flooding stress. Of these, two lipoxygenases, four germin-like protein precursors, three stem 28/31 kDa glycoprotein precursors, and one superoxide dismutase [Cu–Zn] were downregulated. A copper amine oxidase was found to have shifted from the basic to acidic zone following flooding stress. Based on these results, it was confirmed by the lignin staining that the lignification was suppressed in the root of soybean under the flooding stress. These results suggest that the roots and hypocotyls of soybean caused the suppression of lignification through decrease of these proteins by downregulation of reactive oxygen species and jasmonate biosynthesis under flooding stress.

Keywords: Cell wall; Flooding; Lignification; Proteome; Soybean

Strain-dependent expression of metabolic proteins in the mouse hippocampus by Daniela D. Pollak; Narkhyun Bae; Gehan Mostafa; Harald Hoeger (pp. 1451-1462).

Individual mouse strains differ significantly in terms of behavior and cognitive function. Strain-specific variation of metabolic protein levels in the hippocampus among various commonly used mouse strains, however, has not been investigated yet. A proteomic approach based on two-dimensional gel electrophoresis (2-DE) coupled with mass spectrometry [high capacity ion trap (HCT)] has been chosen to address this question by determining strain-dependent levels of metabolic proteins in hippocampal tissue of four inbred and one outbred mouse strain. Statistical analysis of protein spots on 2-DE gels of the individual strains (n = 10) revealed significant strain-dependent differences in densities of 39 spots. Subsequent HCT analysis led to the identification of 22 different metabolic proteins presenting with differential protein levels among the five mouse strains investigated. Among those are proteins concerned with the metabolism of amino acid, nucleic acid, carbohydrate and energy. Moreover, proteins known to play a pivotal role in the processes of learning and memory, such as calcium/calmodulin-dependent protein kinase type II alpha chain, were found to present with significant inter-strain variability, which is also in agreement with our previous reports. Strain-specific protein levels of metabolic proteins in the mouse hippocampus may provide some insight into the molecular underpinnings and genetic determination of strain-dependent neuronal function. Furthermore, data presented herein emphasize the significance of the genetic background for the analysis of metabolic pathways in the hippocampus in wild-type mice as well as in gene-targeting experiments.

Keywords: Hippocampus; Inbred mouse strain; Metabolic protein; Proteomics

Superstoichiometric binding of L-Phe to phenylalanine hydroxylase from Caenorhabditis elegans: evolutionary implications by Marte I. Flydal; Tonje C. Mohn; Angel L. Pey; Jessica Siltberg-Liberles; Knut Teigen; Aurora Martinez (pp. 1463-1475).

Phenylalanine hydroxylase (PAH) catalyzes the hydroxylation of L-Phe to L-Tyr. Dysfunctional PAH results in phenylketonuria and mammalian PAH is therefore highly regulated and displays positive cooperativity for L-Phe (Hill coefficient (h) = 2). L-Phe does not bind to the regulatory ACT domain in full-length tetrameric human PAH and cooperativity is elicited by homotropic binding to the catalytic site (Thórólfsson et al. in Biochemistry 41:7573–7585, 2002). PAH from Caenorhabditis elegans (cePAH) is devoid of cooperativity for L-Phe (h = 0.9), and, as shown in this work, structural analysis reveal an additional L-Phe binding site at the regulatory domain of full-length cePAH. This site involves the GA(S)L/ISRP motifs, which are also found in ACT domains of other L-Phe binding proteins, such as prephenate dehydratase. Isothermal titration calorimetry further demonstrated 2 binding sites per subunit for cePAH versus ~1 for hPAH. Steric occlusion of the regulatory site, notably by residues Lys215/Tyr216 from the adjacent catalytic domain, appears to hinder regulatory binding in full-length hPAH. Accordingly, the humanized mutant Q215K/N216Y of cePAH binds ~1.4 L-Phe/subunit. This mutant also displays high catalytic activity and certain positive cooperativity for L-Phe (h = 1.4). Our results support that the acquisition of positive cooperativity in mammalian forms of PAH is accompanied by a closure of the regulatory L-Phe binding site. Concomitantly, the function of the regulatory ACT domain appears to be adapted from amino acid binding to serving the communication of conformational changes among catalytic subunits.

Keywords: Allosterism; Enzyme regulation; Binding stoichiometry; Prephenate dehydratase; Evolution

Supplementing l-leucine to a low-protein diet increases tissue protein synthesis in weanling pigs by Yulong Yin; Kang Yao; Zhaojin Liu; Min Gong; Zheng Ruan; Dun Deng; Bie Tan; Zhiqiang Liu; Guoyao Wu (pp. 1477-1486).

Recent work with young pigs shows that reducing dietary protein intake can improve gut function after weaning but results in inadequate provision of essential amino acids for muscle growth. Because acute administration of l-leucine stimulates protein synthesis in piglet muscle, the present study tested the hypothesis that supplementing l-leucine to a low-protein diet may maintain the activation of translation initiation factors and adequate protein synthesis in multiple organs of post-weaning pigs. Eighteen 21-day pigs (Duroc × Landrace × Yorkshire) were fed low-protein diets (16.9% crude protein) supplemented with 0, 0.27 or 0.55% l-leucine (total leucine contents in the diets being 1.34, 1.61 or 1.88%, respectively). At 35 days of age, protein synthesis was determined using the [²H] phenylalanine flooding-dose technique. Additionally, total and phosphorylated levels of mammalian target of rapamycin (mTOR), ribosomal protein S6 kinase 1 (S6K1), and eIF4E-binding protein-1 (4E-BP1) were measured in longissimus muscle and liver. Compared with the control group, dietary supplementation with 0.55% l-leucine for 2 weeks increased (P < 0.05): (1) the phosphorylated levels of S6K1 and 4E-BP1; (2) protein synthesis in skeletal muscle, liver, the heart, kidney, pancreas, spleen, and stomach; and (3) daily weight gain by 61%. Dietary supplementation with 0.27% l-leucine enhanced (P < 0.05) protein synthesis in proximal small intestine, kidney and pancreas. These novel findings provide a molecular basis for designing effective nutritional means to increase the efficiency of nutrient utilization for protein accretion in neonates.

Keywords: Food; Weanling pigs; Protein synthesis; Tissues

Evidence for cell cycle-dependent, rapamycin-resistant phosphorylation of ribosomal protein S6 at S240/244 by M. Rosner; M. Hengstschläger (pp. 1487-1492).

The ribosomal protein S6 is essential for the formation of the subunits of higher eukaryotic ribosomes, and S6 heterozygosity leads to early embryonal lethality in mice. S6 is phosphorylated at clustered residues S235/236 and S240/244 upon numerous physiological and pathological stimuli. So far, the S6Kinases, S6K1 and S6K2 are the only proven S6 S240/244 phosphorylating enzymes in mammalian cells. The activity of these S6Kinases is strictly regulated via the mammalian target of rapamycin (mTOR) enzyme complex with raptor, named mTORC1. In time course experiments with the mTORC1 inhibitor rapamycin we here demonstrate rapamycin-resistant phosphorylation of the ribosomal protein S6 at S240/244. Serum-restimulation experiments further demonstrated that this rapamycin-resistant S6 240/244 phosphorylation is induced via serum factors in a cell cycle-dependent manner. Our data allow new insights into the regulation of S6 phosphorylation and provide evidence for the existence of rapamycin-resistant S6 phosphorylating kinase activities.

Keywords: mTOR; Phosphorylation; Rapamycin; S6; S6Kinase

SD-8, a novel therapeutic agent active against multidrug-resistant Gram positive cocci by Biswajit Mishra; Vipul Kumar Srivastava; Rama Chaudhry; Rishi Kumar Somvanshi; Abhay Kumar Singh; Kamaldeep Gill; Ramesh Somvanshi; Ishan Kumar Patro; Sharmistha Dey (pp. 1493-1505).

Anti-bacterial drug resistance is one of the most critical concerns among the scientist worldwide. The novel antimicrobial decapeptide SD-8 is designed and its minimal inhibitory concentration and therapeutic index (TI) was found in the range of 1–8 μg/ml and 45–360, respectively, against major group of Gram positive pathogens (GPP). The peptide was also found to be least hemolytic at a concentration of 180 μg/ml, i.e., nearly 77 times higher than its average effective concentration. The kinetics assay showed that the killing time is 120 min for methicillin-sensitive Staphylococcus aureus (MSSA) and 90 min for methicillin-resistant S. aureus (MRSA). Membrane permeabilization is the cause of peptide antimicrobial activity as shown by the transmission electron microscopy studies. The peptide showed the anti-inflammatory property by inhibiting COX-2 with a K _D and K _i values of 2.36 × 10⁻⁹ and 4.8 × 10⁻⁸ M, respectively. The peptide was also found to be effective in vivo as derived from histopathological observations in a Staphylococcal skin infection rat model with MRSA as causative organism.

Keywords: Multidrug resistant; Antimicrobial peptides; Anti-inflammatory; Surface plasmon resonance; Cyclooxygenase; Histopathology

α-Helix peptides designed from EBV-gH protein display higher antigenicity and induction of monocyte apoptosis than the native peptide by Mauricio Urquiza; Johanna Melo-Cardenas; Tatiana Guevara; Ignacia Echeverria; Isabel C. Rodriguez; Magnolia Vanegas; Mario Amzel; Manuel E. Patarroyo (pp. 1507-1519).

We tested the hypothesis that stabilizing α-helix of Epstein–Barr virus gH-derived peptide 11438 used for binding human cells will increase its biological activity. Non-stable α-helix of peptide 11438 was unfolded in an entropy-driven process, despite the opposing effect of the enthalpy factor. Adding and/or changing amino acids in peptide 11438 allowed the designing of peptides 33207, 33208 and 33210; peptides 33208 and 33210 displayed higher helical content due to a decreased unfolding entropy change as was determined by AGADIR, molecular dynamics and circular dichroism analysis. Peptides 33207, 33208 and 33210 inhibited EBV invasion of peripheral blood mononuclear cells and displayed epitopes more similar to native protein than peptide 11438; these peptides could be useful for detecting antibodies induced by native gH protein since they displayed high reactivity with anti-EBV antibodies. Anti-peptide 33207 antibodies showed higher reactivity with EBV than anti-peptide 11438 antibodies being useful for inducing antibodies against EBV. Anti-peptide 33210 antibodies inhibit EBV invasion of epithelial cells better than anti-peptide 11438 antibodies. Peptide 33210 bound to normal T lymphocytes and Raji cells stronger than peptide 11438 and also induced apoptosis of monocytes and Raji cells but not of normal T cells in a similar way to EBV-gH. Peptide 33210 inhibited the monocytes’ development toward dendritic cells better than EBV and peptide 11438. In conclusion, stabilizing the α-helix in peptides 33208 and 33210 designed from peptide 11438 increased the antigenicity and the ability of the antibodies induced by peptides of inhibiting EBV invasion of host cells.

Keywords: gH; gp85; Entropy; Antibody; Configurational entropy; Dendritic cells

Acute cholesterol depletion leads to net loss of the organic osmolyte taurine in Ehrlich Lettré tumor cells by Kasper Rømer Villumsen; Lars Duelund; Ian Henry Lambert (pp. 1521-1536).

In mammalian cells, the organic osmolyte taurine is accumulated by the Na-dependent taurine transporter TauT and released though the volume- and DIDS-sensitive organic anion channel. Incubating Ehrlich Lettré tumor cells with methyl-β-cyclodextrin (5 mM, 1 h) reduces the total cholesterol pool to 60 ± 5% of the control value. Electron spin resonance data indicate a concomitant disruption of cholesterol-rich micro-domains. Active taurine uptake, cellular taurine content, and cell volume are reduced by 50, 20 and 20% compared to control values, respectively, whereas the passive taurine release is increased 4.5-fold under isotonic conditions following cholesterol depletion. However, taurine release under isotonic conditions is insensitive to DIDS and inhibitors of the volume-regulated anion channel. Uptake and release of meAIB are similarly affected following cholesterol depletion. Kinetic analysis reveals that cholesterol depletion increases TauT’s affinity toward taurine but reduces its maximal transport capacity. Cholesterol depletion has no impact on TauT regulation by protein kinases A and C. Phospholipase A₂ activity, which is required for the activation of volume-sensitive organic anion channel (VSOAC), is increased under isotonic and hypotonic conditions following cholesterol depletion, whereas taurine release under hypotonic conditions is reduced following cholesterol depletion. Hence, acute cholesterol depletion of Ehrlich Lettré cells leads to reduced TauT and VSOAC activities and at the same time increases the release of organic osmolytes via a leak pathway different from the volume-sensitive pathways for amino acids and anions.

Keywords: Phospholipase A₂ activity; Lysophospholipids; Cyclodextrin; TauT; VSOAC; Cell volume regulation

Synthesis and biological activity of novel small peptides with aminophosphonates moiety as NOP receptor ligands by Emilia D. Naydenova; Petar T. Todorov; Polina I. Mateeva; Rositza N. Zamfirova; Nikola D. Pavlov; Simeon B. Todorov (pp. 1537-1543).

The aim of the present study was the synthesis and the biological screening of new analogs of Ac-RYYRWK-NH₂, modified at the N-terminal with 1-[(methoxyphosphono)methylamino]cycloalkanecarboxylic acids. The four newly synthesized ligands for the nociceptin/orphanin FQ (N/OFQ) receptor (NOP) have been prepared by solid-phase peptide synthesis—Fmoc-strategy. These compounds were tested for agonistic activity in vitro on electrically stimulated smooth-muscle preparations isolated from vas deferens of Wistar rats. Our data showed that substitution of Arg at position 1 with aminophosphonates moiety decreased significantly the affinity of ligands to the NOP receptor. Furthermore, the enlargement of the cycle (with 5–8 carbon atoms) additionally diminished both the activity and the selectivity for NOP-receptor.

Keywords: Nociceptin/orphanin FQ; NOP receptor; Nociceptin analogs; SPPS; Aminophosphonates; Rat vas deferens

Expression and purification of antimicrobial peptide CM4 by N^pro fusion technology in E. coli by Xiawei Cheng; Wuguang Lu; Shuangquan Zhang; Peng Cao (pp. 1545-1552).

Antimicrobial peptide CM4 is a small cationic peptide with broad-spectrum activities against bacteria, fungi, and tumor cells. Different strategies have been developed to produce small antibacterial peptides using recombinant techniques. To date, no efforts to obtain large quantities of active recombinant CM4 have been reported. In order to establish a bacterium-based CM4 production system, CM4 was cloned into pET28a and expressed with N^pro mutant (EDDIE) fusion. CM4 expressed as EDDIE are deposited as inclusion bodies. On in vitro refolding by switching from chemotropic to kosmotropic conditions, the fusion partner is released from the C-terminal end of the autoprotease by self-cleavage, leaving CM4 protein with an authentic N terminus. Purified CM4 was separated on Ni²⁺-chelating chromatography column and cation-exchange chromatography column. Mass spectroscopic analysis indicated the protein to be 4132.56 Dalton, which equalled the theoretically expected mass. N-terminal sequencing of CM4 showed the sequence corresponded to the native protein. The recombinant CM4 exhibited the same antimicrobial and anti-tumor activity as reported previously. The expression strategy presented in this study allows convenient high yield and easy purification of recombinant CM4 with native sequences.

Keywords: CM4; EDDIE; Refolding; Optimization; Escherichia coli

Melectin MAPs: the influence of dendrimerization on antimicrobial and hemolytic activity by Petr Niederhafner; Lucie Bednárová; Miloš Buděšínský; Martin Šafařík; Sille Ehala; Jan Ježek; Lenka Borovičková; Vladimír Fučík; Václav Čeřovský; Jiřina Slaninová (pp. 1553-1561).

The recently described antimicrobial peptide melectin (MEP, GFLSILKKVLPKVMAHMK-NH₂) exhibits high antimicrobial activity against Gram-positive and Gram-negative bacteria. Here we describe the synthesis and biological activities of 23 new analogues of MEP. We studied the influence of dimerization and tetramerization (MAP-constructs of MEP) on the antimicrobial and hemolytic activities, as well as the role of Met in positions 14 and 17 of the peptide chain. Oxidation of the Met to Met(O) and Met(O₂) decreases antimicrobial activity of all tested bacteria if the peptide is in the monomeric form, however, only to Staphylococcus aureus if in the form of dimer or tetramer. Dimerization and tetramerization increase the undesirable hemolytic activity of the peptides. Interestingly, substitution of Leu for Val in position 6 leads to the decrease of hemolytic activity. Introduction of the isosteric amino acid Nle into positions 14 or 17 or both leads to slight increase of hemolytic activity under preservation of high antimicrobial activities. Unfortunately, dimerization again leads to an increase of hemolytic activity.

Keywords: Melectin; Antibacterial peptides; Dendrimers; Hemolytic activity; Methionine sulfone; Methionine sulfoxide; Multiple antigen peptides (MAPs)

Identification and origin of Nε-homocysteinyl-lysine isopeptide in humans and mice by Rafał Głowacki; Edward Bald; Hieronim Jakubowski (pp. 1563-1569).

Homocysteine (Hcy) metabolites, Hcy-thiolactone and N-Hcy-proteins, have been linked to the pathology of human cardiovascular and neurodegenerative diseases. Hcy-thiolactone is generated in an error-editing reaction in protein biosynthesis when Hcy is selected in place of methionine by methionyl-tRNA synthetase. N-Hcy-protein, in which Hcy is linked via isopeptide bond to ε-amino group of a protein lysine residue, forms in a post-translational reaction of Hcy-thiolactone with proteins. Here, we identify a novel metabolite, Nε-Hcy-Lys, in human and mouse plasma, and show that this metabolite is elevated in genetic (cystathionine β-synthase deficiency in humans and mice, methylenetetrahydrofolate reductase deficiency in mice) or dietary (high Met diet in mice) deficiencies in Hcy metabolism. We also show that Nε-Hcy-Lys is generated by proteolytic degradation of N-Hcy-protein in mouse liver extracts. Our data indicate that free Nε-Hcy-Lys is an important pathology-related component of Hcy metabolism in humans and mice.

Keywords: Cystathionine β-synthase deficiency; Methylenetetrahydrofolate reductase deficiency; Dietary hyperhomocysteinemia; N-Hcy-protein turnover; Nε-Homocysteinyl-lysine

In vivo effects of APP are not exacerbated by BACE2 co-overexpression: behavioural characterization of a double transgenic mouse model by Garikoitz Azkona; Ditsa Levannon; Yoram Groner; Mara Dierssen (pp. 1571-1580).

Down syndrome, the most common genetic disorder leading to mental retardation, is caused by the presence of all or part of an extra copy of chromosome 21. At relatively early ages, Down syndrome patients develop progressive formation and extracellular aggregation of amyloid-β peptide, considered as one of the causal factors for the pathogenesis of Alzheimer’s disease. This neuropathological hallmark has been attributed to the overexpression of APP but could also be contributed by other HSA21 genes. BACE2 maps to HSA21 and is homologous to BACE1, a β-secretase involved in the amyloidogenic pathway of APP proteolysis, and thus it has been hypothesized that the co-overexpression of both genes could contribute to Alzheimer’s like neuropathology present in Down syndrome. The aim of the present study has been to analyse the impact of the co-overexpression of BACE2 and APP, using a double transgenic mouse model. Double transgenic mice did not present any neurological or sensorimotor alterations, nor genotype-dependent anxiety-like behaviour or age-associated cognitive dysfunction. Interestingly, TgBACE2-APP mice showed deregulation of BACE2 expression levels that were significantly increased with respect to single TgBACE2 mice. Co-overexpression of BACE2 and APP did not increase amyloid-β peptide concentration in brain. Our results suggest that the in vivo effects of APP are not exacerbated by BACE2 co-overexpression but may have some protective effects in specific behavioural and cognitive domains in transgenic mice.

Keywords: Down syndrome; Alzheimer disease; BACE2; APP; Chromosome 21

Amino acid metabolism in the portal-drained viscera of young pigs: effects of dietary supplementation with chitosan and pea hull by Yulong Yin; Ruilin Huang; Tiejun Li; Zheng Ruan; Mingyong Xie; Zeyuan Deng; Yongqing Hou; Guoyao Wu (pp. 1581-1587).

Recent studies indicate extensive catabolism of amino acids (AA) by the portal-drained viscera (PDV) of pigs and humans. Because of ethical concerns over invasive surgical procedures on infants or adults, in vivo investigations are often performed with the pig which is both an agriculturally important livestock species and a widely used animal model for nutritional and physiological studies in humans. Here, we described a new technique for implanting chronic catheters into the portal vein, ileal mesenteric vein, and carotid artery to study AA metabolism in the PDV of young pigs. This method allowed for the reduction of surgery time by 1 h and measurements of the entry of dietary AA into the portal circulation. Using such an approach, we found that dietary supplementation with 100 mg/kg chitosan (a prebiotic and a polysaccharide not digested by animal cells) reduced oxygen consumption, as well as the net absorption of dietary AA into the portal vein, thereby enhancing their bioavailability for extraintestinal tissues. In contrast, opposite results were obtained with dietary supplementation of 12% pea-hull (containing 95% of fermentable nonstarch polysaccharide). Thus, this improved technique is useful to quantify in vivo absorption and metabolism of dietary AA in young pigs.

Keywords: Amino acids; Catheter implantation; Prebiotics; Pigs; Portal vein-drained viscera

Compatibility of the conformationally rigid CF₃-Bpg side chain with the hydrophobic coiled-coil interface by Mario Salwiczek; Pavel K. Mikhailiuk; Sergii Afonin; Igor V. Komarov; Anne S. Ulrich; Beate Koksch (pp. 1589-1593).

3-(Trifluoromethyl)bicyclopent-[1.1.1]-1-yl glycine (CF₃-Bpg) has previously been established as a useful ¹⁹F NMR label to analyse the structures of oligomeric membrane-active peptides or transmembrane segments. To systematically examine the effect of side chain volume, conformational rigidity, and hydrophobicity of CF₃-Bpg in polypeptide environments the amino acid was incorporated into an established coiled-coil based screening system. A single substitution of either valine (position a16) or leucine (position d19) within the hydrophobic core of the heteromeric coiled coil has practically no effect on its structure. Despite its comparatively high hydrophobicity, however, the stiff and bulky side chain of CF₃-Bpg is not so well accommodated by the hydrophobic core as it leads to a more pronounced destabilization than observed for other, more polar fluorinated amino acids which carry more flexible side chains. CF₃-Bpg is therefore a useful ¹⁹F NMR label, though not for monitoring the stability of such helix–helix interactions.

Keywords: Fluorinated amino acids; CF₃-Bicyclopentyl glycine; α-Helical coiled coil; Solid state ¹⁹F NMR; ¹⁹F labelling

Predicting protein–protein interactions from protein sequences using meta predictor by Jun-Feng Xia; Xing-Ming Zhao; De-Shuang Huang (pp. 1595-1599).

A novel method is proposed for predicting protein–protein interactions (PPIs) based on the meta approach, which predicts PPIs using support vector machine that combines results by six independent state-of-the-art predictors. Significant improvement in prediction performance is observed, when performed on Saccharomyces cerevisiae and Helicobacter pylori datasets. In addition, we used the final prediction model trained on the PPIs dataset of S. cerevisiae to predict interactions in other species. The results reveal that our meta model is also capable of performing cross-species predictions. The source code and the datasets are available at http://home.ustc.edu.cn/~jfxia/Meta_PPI.html.

Keywords: Protein–protein interactions; Support vector machine; Meta approach; Protein sequence; Feature representation

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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.39, #5)