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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.44, #2)

The 1,2,4-triazole as a scaffold for the design of ghrelin receptor ligands: development of JMV 2959, a potent antagonist by Aline Moulin; Luc Brunel; Damien Boeglin; Luc Demange; Johanne Ryan; Céline M’Kadmi; Séverine Denoyelle; Jean Martinez; Jean-Alain Fehrentz (pp. 301-314).

Ghrelin is a 28-residue peptide acylated with an n-octanoyl group on the Ser 3 residue, predominantly produced by the stomach. Ghrelin displays strong growth hormone (GH) releasing activity, which is mediated by the activation of the so-called GH secretagogue receptor type 1a (GHS-R1a). Given the wide spectrum of biological activities of Ghrelin in neuroendocrine and metabolic pathways, many research groups, including our group, developed synthetic peptide, and nonpeptide GHS-R1a ligands, acting as agonists, partial agonists, antagonists, or inverse agonists. In this highlight article, we will focus on the discovery of a GHS-R1a antagonist compound, JMV 2959, which has been extensively studied in different in vitro and in vivo models. We will first describe the peptidomimetic approach that led us to discover this compound. Then we will review the results obtained with this compound in different studies in the fields of food intake and obesity, addictive behaviors, hyperactivity and retinopathy.

Keywords: Addiction; Antagonist; Anti-obesity; Ghrelin receptor; Peptidomimetic; 1,2,4-Triazole

Manufacturing of peptides exhibiting biological activity by Aleksandra Zambrowicz; Monika Timmer; Antoni Polanowski; Gert Lubec; Tadeusz Trziszka (pp. 315-320).

Numerous studies have shown that food proteins may be a source of bioactive peptides. Those peptides are encrypted in the protein sequence. They stay inactive within the parental protein until release by proteolytic enzymes (Mine and Kovacs-Nolan in Worlds Poult Sci J 62(1):87–95, 2006; Hartman and Miesel in Curr Opin Biotechnol 18:163–169, 2007). Once released the bioactive peptides exhibit several biofunctionalities and may serve therapeutic roles in body systems. Opioid peptides, peptides lowering high blood pressure, inhibiting platelet aggregation as well as being carriers of metal ions and peptides with immunostimulatory, antimicrobial and antioxidant activities have been described (Hartman and Miesel in Curr Opin Biotechnol 18:163–169, 2007). The biofunctional abilities of the peptides have therefore aroused a lot of scientific, technological and consumer interest with respect to the role of dietary proteins in controlling and influencing health (Möller et al. in Eur J Nutr 47(4):171–182, 2008). Biopeptides may find wide application in food production, the cosmetics industry as well as in the prevention and treatment of various medical conditions. They are manufactured by chemical and biotechnological methods (Marx in Chem Eng News 83(11):17–24. 2005; Hancock and Sahl in Nat Biotechnol 24(12):1551–1557, 2006). Depending on specific needs (food or pharmaceutical industry) different degrees of peptide purifications are required. This paper discusses the practicability of manufacturing bioactive peptides, especially from food proteins.

Keywords: Bioactive peptides; Enzymatic hydrolysis; Immunostimulatory activity; Ovokinin

Direct access to side chain N,N′-diaminoalkylated derivatives of basic amino acids suitable for solid-phase peptide synthesis by Jean-Philippe Pitteloud; Nina Bionda; Predrag Cudic (pp. 321-333).

A simple and efficient one-pot procedure that enables rapid access to orthogonally protected N,N′-diaminoalkylated basic amino acid building blocks fully compatible with standard Boc and Fmoc solid-phase peptide synthesis is reported. Described synthetic approach includes double reductive alkylation of N ^α-protected diamino acids with N-protected amino aldehydes in the presence of sodium cyanoborohydride. This approach allows preparation of symmetrical, as well as unsymmetrical, basic amino acid derivatives with branched side-chains that can be further modified, enhancing their synthetic utility. The suitability of the synthesized branched basic amino acid building blocks for use in standard solid-phase peptide synthesis has been demonstrated by synthesis of an indolicidin analogue in which the lysine residue was substituted with the synthetic derivative N α-(9H-fluorenyl-9-methoxycarbonyl)-N β,N β ′-bis[2-(tert-butoxycarbonylamino)ethyl]-l-2,3-diaminopropionic acid. This substitution resulted in an analogue with more ordered secondary structure in 2,2,2-trifluoroethanol and enhanced antibacterial activity without altering hemolytic activity.

Keywords: N-alkylation; Reductive amination; One-pot procedure; Branched basic amino acids; Solid-phase peptide synthesis; Indolicidin

Synthesis of new β-amidodehydroaminobutyric acid derivatives and of new tyrosine derivatives using copper catalyzed C–N and C–O coupling reactions by G. Pereira; H. Vilaça; P. M. T. Ferreira (pp. 335-344).

Several β-amidodehydroaminobutyric acid derivatives were prepared from N,C-diprotected β-bromodehydroaminobutyric acids and amides by a copper catalyzed C–N coupling reaction. The best reaction conditions include the use of a catalytic amount of CuI, N,N′-dimethylethylenediamine as ligand and K₂CO₃ as base in toluene at 110 °C. The stereochemistry of the products was determined using NOE difference experiments and the results obtained are in agreement with an E-stereochemistry. Thus, the stereochemistry is maintained in the case of the E-isomers of β-bromodehydroaminobutyric acid derivatives, but when the Z-isomers were used as substrates the reaction proceeds with inversion of configuration. The use of β-bromodehydrodipeptides as substrates was also tested. It was found that the reaction outcome depend on the stereochemistry of the β-bromodehydrodipeptide and on the nature of the first amino acid residue. The products isolated were the β-amidodehydrodipeptide derivatives and/or the corresponding dihydropyrazines. The same catalytic system (CuI/N,N′-dimethylethylene diamine) was used in the C–O coupling reactions between a tyrosine derivative and aryl bromides. The new O-aryltyrosine derivatives were isolated in moderate to good yields. The photophysical properties of two of these compounds were studied in four solvents of different polarity. The results show that these compounds after deprotection can be used as fluorescence markers.

Keywords: Copper; N,N′-dimethylethylene diamine; Cross-couplings; Dehydroamino acid derivatives; Fluorescent amino acids

Proteomics analysis of sensitive and tolerant barley genotypes under drought stress by Rehana Kausar; Muhammad Arshad; Armghan Shahzad; Setsuko Komatsu (pp. 345-359).

Drought is a severe environmental constraint to plant productivity and an important factor limiting barley yield. To investigate the initial response of barley to drought stress, changes in protein profile were analyzed using a proteomics technique. Three-day-old barley seedlings of sensitive genotype 004186 and tolerant genotype 004223 were given two treatments, one with 20 % polyethylene glycol and the second with drought induced by withholding water. After 3 days of treatments, proteins were extracted from shoots and separated by 2-dimensional polyacrylamide gel electrophoresis. Metabolism related proteins were decreased in the sensitive genotype under drought; however, they were increased in the tolerant genotype. Photosynthetic related proteins were decreased and increased among the three sensitive and three tolerant genotypes, respectively. In addition, amino acid synthesis and degradation related proteins were increased and decreased among the three tolerant genotypes. These results suggest that chloroplastic metabolism and energy related proteins might play a significant role in the adaptation process of barley seedlings under drought stress.

Keywords: Barley; Proteomics; Drought; Sensitive genotype; Tolerant genotype

Formation of S-(carboxymethyl)-cysteine in rat liver mitochondrial proteins: effects of caloric and methionine restriction by Alba Naudí; Mariona Jové; Daniel Cacabelos; Victoria Ayala; Rosanna Cabre; Pilar Caro; José Gomez; Manuel Portero-Otín; Gustavo Barja; Reinald Pamplona (pp. 361-371).

Maillard reaction contributes to the chemical modification and cross-linking of proteins. This process plays a significant role in the aging process and determination of animal longevity. Oxidative conditions promote the Maillard reaction. Mitochondria are the primary site of oxidants due to the reactive molecular species production. Mitochondrial proteome cysteine residues are targets of oxidative attack due to their specific chemistry and localization. Their chemical, non-enzymatic modification leads to dysfunctional proteins, which entail cellular senescence and organismal aging. Previous studies have consistently shown that caloric and methionine restrictions, nutritional interventions that increase longevity, decrease the rate of mitochondrial oxidant production and the physiological steady-state levels of markers of oxidative damage to macromolecules. In this scenario, we have detected S-(carboxymethyl)-cysteine (CMC) as a new irreversible chemical modification in mitochondrial proteins. CMC content in mitochondrial proteins significantly correlated with that of the lysine-derived analog N ^ε-(carboxymethyl)-lysine. The concentration of CMC is, however, one order of magnitude lower compared with CML likely due in part to the lower content of cysteine with respect to lysine of the mitochondrial proteome. CMC concentrations decreases in liver mitochondrial proteins of rats subjected to 8.5 and 25 % caloric restriction, as well as in 40 and 80 % methionine restriction. This is associated with a concomitant and significant increase in the protein content of sulfhydryl groups. Data presented here evidence that CMC, a marker of Cys-AGE formation, could be candidate as a biomarker of mitochondrial damage during aging.

Keywords: Aging; Carboxymethylated proteins; Dietary restriction; Oxidative stress; Protein damage; Protein cysteine content; Sulfhydryl groups

Transport of l-proline by the proton-coupled amino acid transporter PAT2 in differentiated 3T3-L1 cells by Katja Zebisch; Matthias Brandsch (pp. 373-381).

Mechanism and substrate specificity of the proton-coupled amino acid transporter 2 (PAT2, SLC36A2) have been studied so far only in heterologous expression systems such as HeLa cells and Xenopus laevis oocytes. In this study, we describe the identification of the first cell line that expresses PAT2. We cultured 3T3-L1 cells for up to 2 weeks and differentiated the cells into adipocytes in supplemented media containing 2 μM rosiglitazone. During the 14 day differentiation period the uptake of the prototype PAT2 substrate l-[³H]proline increased ~5-fold. The macro- and microscopically apparent differentiation of 3T3-L1 cells coincided with their H⁺ gradient-stimulated uptake of l-[³H]proline. Uptake was rapid, independent of a Na⁺ gradient but stimulated by an inwardly directed H⁺ gradient with maximal uptake occurring at pH 6.0. l-Proline uptake was found to be mediated by a transport system with a Michaelis constant (K_t) of 130 ± 10 μM and a maximal transport velocity of 4.9 ± 0.2 nmol × 5 min⁻¹mg of protein⁻¹. Glycine, l-alanine, and l-tryptophan strongly inhibited l-proline uptake indicating that these amino acids also interact with the transport system. It is concluded that 3T3-L1 adipocytes express the H⁺-amino acid cotransport system PAT2.

Keywords: 3T3-L1 cells; SLC36A2; Membrane transport; PAT2; Adipocyte

The l-α-amino acid receptor GPRC6A is expressed in the islets of Langerhans but is not involved in l-arginine-induced insulin release by Sanela Smajilovic; Christoffer Clemmensen; Lars Dan Johansen; Petrine Wellendorph; Jens Juul Holst; Peter Grevsen Thams; Egbuna Ogo; Hans Bräuner-Osborne (pp. 383-390).

GPRC6A is a seven-transmembrane receptor activated by a wide range of l-α-amino acids, most potently by l-arginine and other basic amino acids. The receptor is broadly expressed, but its exact physiological role remains to be elucidated. It is well established that l-arginine stimulates insulin secretion; therefore, the receptor has been hypothesized to have a role in regulating glucose metabolism. In this study, we demonstrate that GPRC6A is expressed in islets of Langerhans, but activation of the receptor by l-arginine did not stimulate insulin secretion. We also investigated central metabolic parameters in GPRC6A knockout mice compared with wildtype littermates and found no difference in glucose metabolism or body fat percentage when mice were administered a standard chow diet. In conclusion, our data do not support a role for GPRC6A in l-arginine-induced insulin release and glucose metabolism under normal physiological conditions.

Keywords: GPRC6A; l-Arginine; Insulin release

Mechanism of cysteine-dependent inactivation of aspartate/glutamate/cysteine sulfinic acid α-decarboxylases by Pingyang Liu; Michael P. Torrens-Spence; Haizhen Ding; Bruce M. Christensen; Jianyong Li (pp. 391-404).

Animal aspartate decarboxylase (ADC), glutamate decarboxylase (GDC) and cysteine sulfinic acid decarboxylase (CSADC) catalyze the decarboxylation of aspartate, glutamate and cysteine sulfinic acid to β-alanine, γ-aminobutyric acid and hypotaurine, respectively. Each enzymatic product has been implicated in different physiological functions. These decarboxylases use pyridoxal 5-phosphate (PLP) as cofactor and share high sequence homology. Analysis of the activity of ADC in the presence of different amino determined that beta-alanine production from aspartate was diminished in the presence of cysteine. Comparative analysis established that cysteine also inhibited GDC and CSADC in a concentration-dependent manner. Spectral comparisons of free PLP and cysteine, together with ADC and cysteine, result in comparable spectral shifts. Such spectral shifts indicate that cysteine is able to enter the active site of the enzyme, interact with the PLP-lysine internal aldimine, form a cysteine-PLP aldimine and undergo intramolecular nucleophilic cyclization through its sulfhydryl group, leading to irreversible ADC inactivation. Cysteine is the building block for protein synthesis and a precursor of cysteine sulfinic acid that is the substrate of CSADC and therefore is present in many cells, but the presence of cysteine (at comparable concentrations to their natural substrates) apparently could severely inhibit ADC, CSADC and GDC activity. This raises an essential question as to how animal species prevent these enzymes from cysteine-mediated inactivation. Disorders of cysteine metabolism have been implicated in several neurodegenerative diseases. The results of our study should promote research in terms of mechanism by which animals maintain their cysteine homeostasis and possible relationship of cysteine-mediated GDC and CSADC inhibition in neurodegenerative disease development.

Keywords: Cysteine; Inactivation; PLP-containing decarboxylase; Hypotaurine; β-Alanine; γ-Aminobutyric acid

RALDH2, the enzyme for retinoic acid synthesis, mediates meiosis initiation in germ cells of the female embryonic chickens by Minli Yu; Ping Yu; Imdad H. Leghari; Chutian Ge; Yuling Mi; Caiqiao Zhang (pp. 405-412).

Meiosis is a process unique to the differentiation of germ cells and exhibits sex-specific in timing. Previous studies showed that retinoic acid (RA) as the vitamin A metabolite is crucial for controlling Stra8 (Stimulated by retinoic acid gene 8) expression in the gonad and to initiate meiosis; however, the mechanism by which retinoid-signaling acts has remained unclear. In the present study, we investigated the role of the enzyme retinaldehyde dehydrogenase 2 (RALDH2) which catalyzes RA synthesizes by initiating meiosis in chicken ovarian germ cells. Meiotic germ cells were first detected at day 15.5 in chicken embryo ovary when the expression of synaptonemal complex protein 3 (Scp3) and disrupted meiotic cDNA 1 homologue (Dmc1) became elevated, while Stra8 expression was specifically up-regulated at day 12.5 before meiosis onset. It was observed from the increase in Raldh2 mRNA expression levels and decreases in Cyp26b1 (the enzyme for RA catabolism) expression levels during meiosis that requirement for RA accumulation is essential to sustain meiosis. This was also revealed by RA stimulation of the cultured ovaries with the initiation of meiosis response, and the knocking down of the Raldh2 expression during meiosis, leading to abolishment of RA-dependent action. Altogether, these studies indicate that RA synthesis by the enzyme RALDH2 and signaling through its receptor is crucial for meiosis initiation in chicken embryonic ovary.

Keywords: Chicken; Meiosis initiation; Ovary; RALDH2; Retinoic acid; shRNA interference

The involvement of AMPA–ERK1/2–BDNF pathway in the mechanism of new antidepressant action of prokinetic meranzin hydrate by Ying Xie; Xi Huang; Sui-yu Hu; Ying-jin Zhang; Yang Wang; Xin-jian Qiu; Ping Ren; Rong Fan; Chun-hu Zhang; Wei-bin Xie; Hui Ji; Juan He; Xiao Chen; Linhong Xie; Zhao-qian Liu; Hong-hao Zhou (pp. 413-422).

It was recently discovered that ketamine can relieve depression in a matter of hours through an action on α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. This is much more rapid than the several weeks required for the available antidepressants to show therapeutic efficacy. However, ketamine has negative side effects. The aim of this study was to determine whether the natural prokinetic drug meranzin hydrate (MH) has a fast-acting antidepressant effect mediated by AMPA receptors. By means of in vivo and in vitro experiments, we found that (1) treatment of rats with MH at 9 mg/kg decreased immobility time in a forced swimming test (FST), as did the popular antidepressant fluoxetine and the AMPA receptor positive modulator aniracetam. Pretreatment of rats with NBQX (10 mg/kg), an antagonist of AMPA receptors, blocked this effect of MH. (2) MH increased number of crossings of forced swimming rats in the open field test. (3) FST enhanced hippocampal ERK1/2, p-ERK1/2 and BDNF expression levels. MH (9 mg/kg) treatment further up-regulated hippocampal p-ERK1/2 and BDNF expression levels, and this effect was prevented by NBQX. (4) MH-increased BDNF expression corresponded with MH-decreased immobility time in the FST. (5) In vitro experiments, we found that incubation of rats hippocampus slices with MH (10, 20 μM respectively) increased concentrations of BDNF and p-ERK1/2. This effect of MH (20 μM) were prevented by NBQX. In conclusion, in animals subjected to acute stress, the natural prokinetic drug MH produced a rapid effect mediated by AMPA receptors and involving BDNF modulation through the ERK1/2 pathway.

Keywords: MH; Forced swimming test; BDNF; Glutamate; AMPA; ERK1/2

Differential topochemistry of three cationic amino acid transporter proteins, hCAT1, hCAT2 and hCAT3, in the adult human brain by K. Jäger; S. Wolf; H. Dobrowolny; J. Steiner; H. Nave; E. Maronde; B. Bogerts; H.-G. Bernstein (pp. 423-433).

The cellular uptake of l-arginine and other cationic amino acids (such as l-lysine and l-ornithine) is mainly mediated by cationic amino acid transporter (CAT) proteins. Despite the important roles of cationic amino acid transporters for normal brain functioning and various brain diseases there is currently only fragmentary knowledge about their cellular and regional distribution patterns in the human brain. We mapped the immunohistochemical localization of human cationic amino acid transporters 1, 2 and 3 (hCAT1, 2, and 3) throughout five adult human brains and found a wide but uneven distribution of these transporters. All three hCAT1s were mainly localized in neurons, but were also found in numerous astrocytes, oligodendrocytes, plexus choroideus epithelial cells, and small blood vessels. The highest density of hCAT expressing neurons was observed in the hypothalamus, in some areas of the cerebral cortex, the thalamic reticular nucleus and the caudate nucleus, whereas weak to moderate expression was detected in the hippocampus, the prefrontal cortex (hCAT1 only), pons, brain stem and cerebellum. In contrast to what has been found in rodent brain, we detected hCAT2 and hCAT3 also in astrocytes. Overall, each hCAT has its characteristic, individual cerebral expression patterns, which, however, overlap with the others.

Keywords: hCAT1; hCAT2; hCAT3; Human brain; Distribution; Immunohistochemistry

A practical route to long-chain non-natural α,ω-diamino acids by Giuseppina Brasile; Laura Mauri; Sandro Sonnino; Federica Compostella; Fiamma Ronchetti (pp. 435-441).

An efficient method for the synthesis of long-chain α,ω-diamino acids, starting from natural α-amino acids, has been developed. The long-chain skeleton has been generated through condensation between a protected aldehyde, derived from l-aspartic acid, and an ylide obtained from an ω-hydroxy-alkyl phosphonium salt. After conversion of the ω-hydroxy group into an amine, catalytic hydrogenation produced the N,N′-protected α,ω-diamino acid. The present route to α,ω-diamino acids allows the modulation of the chain length depending on the length of the ylide used for the Wittig olefination reaction.

Keywords: Lipidic α-amino acids (LAAs); Unnatural fatty acids; Wittig olefination; α,ω-Diamino acids

Novel thiol- and thioether-containing amino acids: cystathionine and homocysteine families by Luigi Longobardo; Nunzia Cecere; Marina DellaGreca; Ivan de Paola (pp. 443-448).

Natural l-homocysteine and l,l-cystathionine, along with a series of unnatural analogues, have been prepared from l-aspartic and l-glutamic acid. Manipulation of the protected derivatives provided ω-iodoamino acids, which were used in thioalkylation reactions of sulfur nucleophiles, such as the ester of l-cysteine and potassium thioacetate.

Keywords: Cystathionine; Homocysteine; Cysteine thioalkylation; Bis-amino acids; Unnatural amino acids

Synthesis of new poly(ether–urethane–urea)s based on amino acid cyclopeptide and PEG: study of their environmental degradation by Fatemeh Rafiemanzelat; Abolfazl Fathollahi Zonouz; Giti Emtiazi (pp. 449-459).

Conventional polyurethanes (PUs) are among biomaterials not intended to degrade but are susceptible to hydrolytic, oxidative and enzymatic degradation in vivo. Biodegradable PUs are typically prepared from polyester polyols, aliphatic diisocyanates and chain extenders. In this work we have developed a degradable monomer based on α-amino acid to accelerate hard segment degradation. Thus a new class of degradable poly(ether–urethane–urea)s (PEUUs) was synthesized via direct reaction of 4,4′-methylene-bis(4-phenylisocyanate) (MDI), l-leucine anhydride (LA) and polyethylene glycol with molecular weight of 1,000 (PEG-1000) as polyether soft segment. The resulting polymers are environmentally biodegradable and thermally stable. Decomposition temperatures for 5 % weight loss occurred above 300 °C by TGA in nitrogen atmospheres. Some structural characterization and physical properties of these polymers before and after degradation in soil, river water and sludge are reported. The environmental degradation of the polymer films was investigated by SEM, FTIR, TGA, DSC, GPC and XRD techniques. A significant rate of degradation occurred in PEUU samples under river water and sludge condition. The polymeric films were not toxic to E. coli (Gram negative), Staphylococcus aureus and Micrococcus (Gram positive) bacteria and showed good biofilm formation on polymer surface. Our results show that hard segment degraded selectively as much as soft segment and these polymers are susceptible to degradation in soil and water. Thus our study shows that new environment-friendly polyurethane, which can degrade in soil, river water and sludge, is synthesized.

Keywords: Biodegradable segmented copolyurethane; PEG; Amino acid anhydride; Cell-toxicity

TAT-CC fusion protein depresses the oncogenicity of BCR-ABL in vitro and in vivo through interrupting its oligomerization by Zheng-Lan Huang; Miao Gao; Mao-Sheng Ji; Kun Tao; Qing Xiao; Liang Zhong; Jian-Ming Zeng; Wen-Li Feng (pp. 461-472).

Chronic myeloid leukemia (CML) is a clonal hematologic malignancy characterized by the BCR-ABL protein. BCR-ABL is a constitutively active tyrosine kinase and plays a critical role in the pathogenesis of CML. Imatinib mesylate, a selective tyrosine kinase inhibitor, is effective in CML, but drug resistance and relapse occur. The coiled-coil (CC) domain located in BCR_1–72 mediates BCR-ABL tetramerization, which is essential for the activation of tyrosine kinase and transformation potential of BCR-ABL. CC domain is supposed to be a therapeutic target for CML. We purified a TAT-CC protein competively binding with the endogenous CC domain to reduce BCR-ABL kinase activity. We found that TAT-CC co-located and interacted with BCR-ABL in Ba/F3-p210 and K562 cells. It induced apoptosis and inhibited proliferation in these cells. It increased the sensitivity of these cells to imatinib and reduced the phosphorylation of BCR-ABL, CRKL and STAT5. We confirmed that TAT-CC could attenuate the oncogenicity of Ba/F3-p210 cells and diminish the volume of K562 solid tumor in mice. We conclude targeting the CC may provide a complementary therapy to inhibit BCR-ABL oncogenicity.

Keywords: TAT; Coiled-coil domain; BCR-ABL; Oligomerization; Oncogenicity

Altering the Tat-derived peptide bioactivity landscape by changing the arginine side chain length by Cheng-Hsun Wu; Yi-Ping Chen; Chung-Yuan Mou; Richard P. Cheng (pp. 473-480).

Mutations of proteins with dual activities that lead to enhancement of one activity are frequently accompanied by attenuation of the other activity. However, this mutational negative trade-off phenomenon typically only involves the canonical 20 amino acids. To test the effect of non-canonical amino acids on the negative trade-off phenomenon, two bioactivities of HIV-1 Tat-derived peptides were monitored upon changing the Arg side chain length. In contrast to the expected mutational negative trade-off, shortening Arg by one methylene resulted in both higher TAR RNA binding specificity and higher cellular uptake. These results suggest that introducing previously unexploited building blocks, even if the difference is only one methylene, can alter the peptide bioactivity landscape leading to the enhancement of multiple bioactivities.

Keywords: Arginine; Side chain length; Cellular uptake; RNA recognition

Antimicrobial peptide diversity in the skin of the torrent frog, Amolops jingdongensis by Xiaoqin He; Shilong Yang; Lin Wei; Rui Liu; Ren Lai; Mingqiang Rong (pp. 481-487).

Antimicrobial peptide diversity has been found in some amphibians. The diversity of antimicrobial peptides may have resulted from the diversity of microorganisms encountered by amphibians. Peptidomics and genomics analyses were used to study antimicrobial peptide diversity in the skin secretions of the torrent frog, Amolops jingdongensis. Thirty-one antimicrobial peptides belonging to nine groups were identified in the skin secretions of this frog. Among them, there are two novel antimicrobial groups (jingdongin-1 and -2) with unique structural motifs. The other seven groups belong to known antimicrobial peptide families, namely brevinin-1, brevinin-2, odorranain-F, esculentin-2, temporin, amolopin-3, and ranacyclin. Combined with previous reports, more than 13 antimicrobial peptide groups have been identified from the genus Amolops. Most of these antimicrobial peptide groups are also found in amphibians belonging to the genus Rana or Odorrana which suggests a possible evolutionary connection among Amolops, Rana, and Odorrana. Two novel antimicrobial groups (jingdongin-1 and -2) were synthesized and their antimicrobial activities were assayed. Some of them showed strong antimicrobial abilities against microorganisms including Gram-negative and -positive bacteria, and fungi. The extreme diversity of antimicrobial peptides in the Amolops amphibians was demonstrated. In addition, several novel peptide templates were provided for antimicrobial agent design.

Keywords: Antimicrobial peptides; Diversity; Amphibian; Skin; Amolops

Principal component analysis of the relationship between the d-amino acid concentrations and the taste of the sake by Kaori Okada; Yoshitaka Gogami; Tadao Oikawa (pp. 489-498).

We performed sensory evaluations on 141 bottles of sake and analyzed the relationship between the d-amino acid concentrations, and the taste of the sake using principal component analysis, which yielded seven principal components (PC1–7) that explained 100 % of the total variance in the data. PC1, which explains 33.6 % of the total variance, correlates most positively with strong taste and most negatively with balanced tastes. PC2, which explains 54.4 % of the total variance, correlates most positively with a sweet taste and most negatively with bitter and sour tastes. Sakes brewed with “Kimoto yeast starter” and “Yamahaimoto” had high scores for PC1 and PC2, and had strong taste in comparison with sakes brewed with “Sokujo-moto”. When present at concentrations below 50 μM, d-Ala did not affect the PC1 score, but all the sakes showed a high PC1 score, when the d-Ala was above 100 μM. Similar observations were found for the d-Asp and d-Glu concentrations with regard to PC1, and the threshold concentrations of d-Asp and d-Glu that affected the taste were 33.8 and 33.3 μM, respectively. Certain bacteria present in sake, especially lactic acid bacteria, produce d-Ala, d-Asp and d-Glu during storage, and these d-amino acids increased the PC1 score and produced a strong taste (Nojun). When d- and l-Ala were added to the sakes, the value for the umami taste in the sensory evaluation increased, with the effect of d-Ala being much stronger than that of l-Ala. The addition of 50–5,000 μM dl-Ala did not effect on the aroma of the sakes at all.

Keywords: d-Amino acid; Taste of sake; Principal component analysis

Antitumor activity of novel chimeric peptides derived from cyclinD/CDK4 and the protein transduction domain 4 by Haili Wang; Xi Chen; Yanping Chen; Lei Sun; Guodong Li; Mingxia Zhai; Wenjie Zhai; Qiaozhen Kang; Yanfeng Gao; Yuanming Qi (pp. 499-510).

CyclinD1/CDK4 and cyclinD3/CDK4 complexes are key regulators of the cell progression and therefore constitute promising targets for the design of anticancer agents. In the present study, the key peptide motifs were selected from these two complexes. Chimeric peptides with these peptides conjugated to the protein transduction domain 4 (PTD4) were designed and synthesized. The chimeric peptides, PTD4-D1, PTD4-D3, PTD4-K4 exhibited significant anti-proliferation effects on cancer cell lines. These peptides could compete with the cyclinD/CDK4 complex and induce the G1/S phase arrest and apoptosis of cancer cells. In the tumor challenge experiment, these peptides showed potent antitumor effects with no significant side effects. Our results suggested that these peptides could be served as novel leading compounds with potent antitumor activity.

Keywords: CyclinD; CDK4; Peptide; Antitumor; Apoptosis

Synthesis of a conformationally constrained δ-amino acid building block by Elaine O’Reilly; Lara Pes; Yannick Ortin; Helge Müller-Bunz; Francesca Paradisi (pp. 511-518).

Conformationally restricted amino acids are important components in peptidomimetics and drug design. Herein, we describe the synthesis of a novel, non-proteinogenic constrained delta amino acid containing a cyclobutane ring, cis-3(aminomethyl)cyclobutane carboxylic acid (ACCA). The synthesis of the target amino acid was achieved in seven steps, with the key reaction being a base induced intramolecular nucleophilic substitution. A small library of dipeptides was prepared through the coupling of ACCA with proteinogenic amino acids.

Keywords: Non-proteinogenic amino acid; Dipeptides; Conformational restriction; Cyclobutane ring

Dietary l-leucine and l-alanine supplementation have similar acute effects in the prevention of high-fat diet-induced obesity by Anne Freudenberg; Klaus J. Petzke; Susanne Klaus (pp. 519-528).

High-protein diets have been shown to alleviate detrimental effects of high-fat diets and this effect can be partially mimicked by dietary l-leucine supplementation. Here, we aimed to elucidate the early mechanisms and the specificity of leucine effects. We performed a 1-week trial with male C57BL/6 mice fed ad libitum with semisynthetic high-fat diets containing an adequate (10 % w/w, AP) or high (50 % w/w, HP) amount of whey protein, or supplemented with l-leucine corresponding to the leucine content within the HP diet (Leu) or supplemented with equimolar l-alanine (Ala). Food and water intake were monitored continuously using a computer-controlled monitor system and body composition changes were assessed using quantitative NMR. HP completely prevented the AP-induced accumulation of body fat. Leu and Ala resulted in a similar reduction of body fat accumulation which was intermediate between AP and HP. There were no significant effects on plasma glucose or insulin. Triacylglycerol content and gene expression of lipogenesis enzymes in liver as well as plasma cholesterol were reduced by HP compared to AP with Leu and Ala again showing intermediate effects. Body fat gain and liver triacylglycerols were strongly correlated with total energy intake. Water intake was rapidly increased by HP feeding and total water intake correlated strongly with total amino nitrogen intake. We concluded that the positive effects of high-protein diets on metabolic syndrome associated traits are acutely due to effects on satiety possibly linked to amino nitrogen intake and on the subsequent suppression of liver lipogenesis without evidence for a specific leucine effect.

Keywords: High-protein diet; l-Leucine supplementation; l-Alanine supplementation; Satiety; Energy intake; Water intake; Metabolic syndrome; Diet-induced fatty liver

Preparation of 3-bromo-l-tyrosine and 3,5-dibromo-l-tyrosine by Robert S. Phillips; Susan Busby; Leia Edenfield; Kevin Wickware (pp. 529-532).

l-Tyrosine is converted to 3-bromo-l-tyrosine in good yield by reaction with 1.2 equiv. of DMSO in HBr/AcOH, while reaction with 2.2 equiv. of DMSO under comparable conditions results in formation of 3,5-dibromo-l-tyrosine in good yield. This is the simplest, safest and most efficient method for the preparation of gram quantities of either 3-bromo-l-tyrosine or 3,5-dibromo-l-tyrosine.

Keywords: Bromination; Bromotyrosine; Dibromotyrosine; DMSO/HBr; Tyrosine

Modulation of taurine release in glucose-free media by glutamate receptors in hippocampal slices from developing and adult mice by Simo S. Oja; Pirjo Saransaari (pp. 533-542).

Taurine has been thought to protect neural cells against cell-damaging conditions to which the hippocampus is particularly vulnerable. We studied now how the release of preloaded [³H]taurine is regulated by glutamate receptors in glucose-free media in slices prepared from the mouse hippocampus from developing (7 days old) and young adult (3 months old) mice, using a superfusion system. The lack of glucose enhanced taurine release more from slices from developing mice than from slices from adults. At both ages ionotropic glutamate agonists significantly increased the release in a receptor-mediated manner. Of the metabotropic glutamate receptors those belonging to the group III were effective. The release was enhanced in adult mice but attenuated in developing mice. Both effects were blocked by the receptor antagonists. The results show that glutamate receptors affect taurine release in the absence of glucose in which condition taurine should be neuroprotective.

Keywords: Taurine release; Glutamate receptors; Hippocampus; Tissue slices; Adults; Developing mice

Evaluation of a technetium-99m labeled bombesin homodimer for GRPR imaging in prostate cancer by Zilin Yu; Giuseppe Carlucci; Hildo J. K. Ananias; Rudi A. J. O. Dierckx; Shuang Liu; Wijnand Helfrich; Fan Wang; Igle J. de Jong; Philip H. Elsinga (pp. 543-553).

Multimerization of peptides can improve the binding characteristics of the tracer by increasing local ligand concentration and decreasing dissociation kinetics. In this study, a new bombesin homodimer was developed based on an ε-aminocaproic acid-bombesin(7–14) (Aca-bombesin(7–14)) fragment, which has been studied for targeting the gastrin-releasing peptide receptor (GRPR) in prostate cancer. The bombesin homodimer was conjugated to 6-hydrazinopyridine-3-carboxylic acid (HYNIC) and labeled with ^99mTc for SPECT imaging. The in vitro binding affinity to GRPR, cell uptake, internalization and efflux kinetics of the radiolabeled bombesin dimer were investigated in the GRPR-expressing human prostate cancer cell line PC-3. Biodistribution and the GRPR-targeting potential were evaluated in PC-3 tumor-bearing athymic nude mice. When compared with the bombesin monomer, the binding affinity of the bombesin dimer is about ten times lower. However, the ^99mTc labeled bombesin dimer showed a three times higher cellular uptake at 4 h after incubation, but similar internalization and efflux characters in vitro. Tumor uptake and in vivo pharmacokinetics in PC-3 tumor-bearing mice were comparable. The tumor was visible on the dynamic images in the first hour and could be clearly distinguished from non-targeted tissues on the static images after 4 h. The GRPR-targeting ability of the ^99mTc labeled bombesin dimer was proven in vitro and in vivo. This bombesin homodimer provides a good starting point for further studies on enhancing the tumor targeting activity of bombesin multimers.

Keywords: GRPR; Bombesin homodimer; Radiolabeled; Imaging; Prostate cancer; PC-3; ^99mTc; HYNIC; SPECT

The effect of acute taurine ingestion on 3-km running performance in trained middle-distance runners by Thomas G. Balshaw; Theodoros M. Bampouras; Timothy J. Barry; S. Andy Sparks (pp. 555-561).

Limited research examining the effect of taurine (TA) ingestion on human exercise performance exists. The aim of this study was to investigate the effect of acute ingestion of 1,000 mg of TA on maximal 3-km time trial (3KTT) performance in trained middle-distance runners (MDR). Eight male MDR (mean ± SD: age 19.9 ± 1.2 years, body mass 69.4 ± 6.6 kg, height 180.5 ± 7.5 cm, 800 m personal best time 121.0 ± 5.3 s) completed TA and placebo (PL) trials 1 week apart in a double-blind, randomised, crossover designed study. Participants consumed TA or PL in capsule form on arrival at the laboratory followed by a 2-h ingestion period. At the end of the ingestion period, participants commenced a maximal simulated 3KTT on a treadmill. Capillary blood lactate was measured pre- and post-3KTT. Expired gas, heart rate (HR), ratings of perceived exertion (RPE), and split times were measured at 500-m intervals during the 3KTT. Ingestion of TA significantly improved 3KTT performance (TA 646.6 ± 52.8 s and PL 658.5 ± 58.2 s) (p = 0.013) equating to a 1.7 % improvement (range 0.34–4.24 %). Relative oxygen uptake, HR, RPE and blood lactate did not differ between conditions (p = 0.803, 0.364, 0.760 and 0.302, respectively). Magnitude-based inference results assessing the likeliness of a beneficial influence of TA were 99.3 %. However, the mechanism responsible for this improved performance is unclear. TA’s potential influence on exercise metabolism may involve interaction with the muscle membrane, the coordination or the force production capability of involved muscles. Further research employing more invasive techniques may elucidate TA’s role in improving maximal endurance performance.

Keywords: Oxygen uptake; Ergogenic aids; Time trial; Endurance running

Comparative efficiency of microbial enzyme preparations versus pancreatin for in vitro alimentary protein digestion by Mireille Andriamihaja; Alain Guillot; Allan Svendsen; Joerg Hagedorn; Sahondra Rakotondratohanina; Daniel Tomé; François Blachier (pp. 563-572).

Utilisation of microbial enzymes may represent an alternative strategy to the use of conventional pancreatin obtained from pig pancreas for the treatment of severe pancreatic insufficiency. In this study, we focused on the capacity of two microbial preparations for their capacity to digest alimentary proteins (caseins and soya proteins) in comparison with pancreatin. These microbial enzymatic preparations were found to be able to generate small, medium-size and larger polypeptides from caseins and soya proteins but were inactivated at pH 3.0. As determined by Liquid Chromatography–Mass Spectrometry analysis, microbial enzymes generated very different peptides from caseins when compared with peptides generated through pancreatin action. These microbial preparations were characterised by relatively low trypsin- and low carboxypeptidase-like activities but high chymotrypsin-like activities and strong capacity for cleavage of caseins at the methionine sites. Although the efficiency of these microbial preparations to increase the rate of absorption of nitrogen-containing compounds in severe pancreatic insufficiency remains to be tested in vivo, our in vitro data indicate proteolytic capacities of such preparations for alimentary protein digestion.

Keywords: Proteins; Digestion; Pancreatin; Microbial enzymes

A similarity distance of diversity measure for discriminating mesophilic and thermophilic proteins by Yong-Chun Zuo; Wei Chen; Guo-Liang Fan; Qian-Zhong Li (pp. 573-580).

The successful prediction of thermophilic proteins is useful for designing stable enzymes that are functional at high temperature. We have used the increment of diversity (ID), a novel amino acid composition-based similarity distance, in a 2-class K-nearest neighbor classifier to classify thermophilic and mesophilic proteins. And the KNN-ID classifier was successfully developed to predict the thermophilic proteins. Instead of extracting features from protein sequences as done previously, our approach was based on a diversity measure of symbol sequences. The similarity distance between each pair of protein sequences was first calculated to quantitatively measure the similarity level of one given sequence and the other. The query protein is then determined using the K-nearest neighbor algorithm. Comparisons with multiple recently published methods showed that the KNN-ID proposed in this study outperforms the other methods. The improved predictive performance indicated it is a simple and effective classifier for discriminating thermophilic and mesophilic proteins. At last, the influence of protein length and protein identity on prediction accuracy was discussed further. The prediction model and dataset used in this article can be freely downloaded from http://wlxy.imu.edu.cn/college/biostation/fuwu/KNN-ID/index.htm .

Keywords: Thermophilic protein; Increment of diversity; K-nearest neighbor; Amino acids; Prediction performance

Characterizing circular peptides in mixtures: sequence fragment assembly of cyclotides from a violet plant by MALDI-TOF/TOF mass spectrometry by Hossein Hashempour; Johannes Koehbach; Norelle L. Daly; Alireza Ghassempour; Christian W. Gruber (pp. 581-595).

Cyclotides are a very abundant class of plant peptides that display significant sequence variability around a conserved cystine-knot motif and a head-to-tail cyclized backbone conferring them with remarkable stability. Their intrinsic bioactivities combined with tools of peptide engineering make cyclotides an interesting template for the design of novel agrochemicals and pharmaceuticals. However, laborious isolation and purification prior to de novo sequencing limits their discovery and hence their use as scaffolds for peptide-based drug development. Here we extend the knowledge about their sequence diversity by analysing the cyclotide content of a violet species native to Western Asia and the Caucasus region. Using an experimental approach, which was named sequence fragment assembly by MALDI-TOF/TOF, it was possible to characterize 13 cyclotides from Viola ignobilis, whereof ten (vigno 1–10) display previously unknown sequences. Amino acid sequencing of various enzymatic digests of cyclotides allowed the accurate assembly and alignment of smaller fragments to elucidate their primary structure, even when analysing mixtures containing multiple peptides. As a model to further dissect the combinatorial nature of the cyclotide scaffold, we employed in vitro oxidative refolding of representative vigno cyclotides and confirmed the high dependency of folding yield on the inter-cysteine loop sequences. Overall this work highlights the immense structural diversity and plasticity of the unique cyclotide framework. The presented approach for the sequence analysis of peptide mixtures facilitates and accelerates the discovery of novel plant cyclotides.

Keywords: Viola ignobilis ; Circular; Cystine-knot; Oxidative folding; Vigno; Peptidomics

Genome-wide computational identification of bicistronic mRNA in humans by Yiming Lu; Yanchun Zhang; Xingyi Hang; Wubin Qu; Gert Lubec; Changsheng Chen; Chenggang Zhang (pp. 597-606).

Mammalian bicistronic mRNA is a recently discovered mammalian gene structure. Several reported cases of mammalian bicistronic mRNA indicated that genes of this structure play roles in some important biological processes. However, a genome-wide computational identification of bicistronic mRNA in mammalian genome, such as human genome, is still lacking. Here we used a comparative genomics approach to identify the frequency of human bicistronic mRNA. We then validated the result by using a new support vector machine (SVM) model. We identified 43 human bicistronic mRNAs in 30 distinct genes. Our literature analysis shows that our method recovered 100 % (6/6) of the previously known bicistronic mRNAs which had been experimentally confirmed by other groups. Our graph theory-based analysis and GO analysis indicated that human bicistronic mRNAs are prone to produce different yet closely functionally related proteins. In addition, we also described and analyzed three different mechanisms of ORF fusion. Our method of identifying bicistronic mRNAs in human genome provides a model for the computational identification of characteristic gene structures in mammalian genomes. We anticipate that our data will facilitate further molecular characterization and functional study of human bicistronic mRNA.

Keywords: Bicistronic mRNA; Computational identification; Domain–domain interaction; Open reading frame fusion; Support vector machine

The impact of either 4-R-hydroxyproline or 4-R-fluoroproline on the conformation and SH3_m-cort binding of HPK1 proline-rich peptide by Andrea Borgogno; Paolo Ruzza (pp. 607-614).

SH3 domains are probably the most abundant molecular-recognition modules of the proteome. A common feature of these domains is their interaction with ligand proteins containing Pro-rich sequences. Crystal and NMR structures of SH3 domains complexes with Pro-rich peptides show that the peptide ligands are bound over a range of up to seven residues in a PPII helix conformation. Short proline-rich peptides usually adopt little or no ordered secondary structure before binding interactions, and consequently their association with the SH3 domain is characterized by unfavorable binding entropy due to a loss of rotational freedom on forming the PPII helix. With the aim to stabilize the PPII helix conformation into the proline-rich decapeptide PPPLPPKPKF (P2), we replaced some proline residues either with the 4(R)-4-fluoro-l-proline (FPro) or the 4(R)-4-hydroxy-l-proline (Hyp). The interactions of P2 analogues with the SH3 domain of cortactin (SH3_m-cort) were analyzed by circular dichroism spectroscopy, while CD thermal transition experiments have been used to determine their propensity to adopt a PPII helix conformation. Results show that the introduction of three residues of Hyp efficiently stabilizes the PPII helix conformation, while it does not improve the affinity towards the SH3 domain, suggesting that additional forces, e.g., electrostatic interactions, are involved in the SH3_m-cort substrate recognition.

Keywords: Cortactin; Pro-rich peptides; 4-R-Fluoroproline; 4-R-Hydroxyproline; Protein–protein interaction

Taurine homeostasis requires de novo synthesis via cysteine sulfinic acid decarboxylase during zebrafish early embryogenesis by Yen-Chia Chang; Shih-Torng Ding; Yen-Hua Lee; Ya-Ching Wang; Ming-Feng Huang; I-Hsuan Liu (pp. 615-629).

Cysteine sulfinic acid decarboxylase (Csad) is the rate-limiting enzyme in the de novo biosynthesis of taurine. There are a number of physiological roles of taurine, such as bile salt synthesis, osmoregulation, lipid metabolism, and oxidative stress inhibition. To investigate the role of de novo synthesis of taurine during embryonic development, zebrafish csad was cloned and functionally analyzed. Semi-quantitative RT-PCR showed that csad transcripts are maternally deposited, while whole-mount in situ hybridization demonstrated that csad is expressed in yolk syncytial layer and various embryonic tissues such as notochord, brain, retina, pronephric duct, liver, and pancreas. Knockdown of csad significantly reduced the embryonic taurine level, and the affected embryos had increased early mortality and cardiac anomalies. mRNA coinjection and taurine supplementation rescued the cardiac phenotypes suggesting that taurine originating from the de novo synthesis pathway plays a role in cardiac development. Our findings indicated that the de novo synthesis pathway via Csad plays a critical role in taurine homeostasis and cardiac development in zebrafish early embryos.

Keywords: Cysteine sulfinic acid decarboxylase; Csad; Taurine; Zebrafish; Embryogenesis

eIF5A dimerizes not only in vitro but also in vivo and its molecular envelope is similar to the EF-P monomer by Camila Arnaldo Olhê Dias; Wanius Garcia; Cleslei Fernando Zanelli; Sandro Roberto Valentini (pp. 631-644).

The protein eukaryotic initiation factor 5A (eIF5A) is highly conserved among archaea and eukaryotes, but not in bacteria. Bacteria have the elongation factor P (EF-P), which is structurally and functionally related to eIF5A. eIF5A is essential for cell viability and the only protein known to contain the amino acid residue hypusine, formed by post-translational modification of a specific lysine residue. Although eIF5A was initially identified as a translation initiation factor, recent studies strongly support a function for eIF5A in the elongation step of translation. However, the mode of action of eIF5A is still unknown. Here, we analyzed the oligomeric state of yeast eIF5A. First, by using size-exclusion chromatography, we showed that this protein exists as a dimer in vitro, independent of the hypusine residue or electrostatic interactions. Protein–protein interaction assays demonstrated that eIF5A can form oligomers in vitro and in vivo, in an RNA-dependent manner, but independent of the hypusine residue or the ribosome. Finally, small-angle X-ray scattering (SAXS) experiments confirmed that eIF5A behaves as a stable dimer in solution. Moreover, the molecular envelope determined from the SAXS data shows that the eIF5A dimer is L-shaped and superimposable on the tRNA^Phe tertiary structure, analogously to the EF-P monomer.

Keywords: eIF5A; Dimer; Hypusine; EF-P; tRNA

Effect of repetitive lysine–tryptophan motifs on the bactericidal activity of antimicrobial peptides by Ramamourthy Gopal; Chang Ho Seo; Peter I. Song; Yoonkyung Park (pp. 645-660).

Previous studies identified lysine- and tryptophan-rich sequences within various cationic antimicrobial peptides. In the present study, we synthesized a series of peptides composed of lysine (K)-tryptophan (W) repeats (KW) n (where n equals 2, 3, 4 or 5) with amidation of the C-terminal to increase cationicity. We found that increases in chain length up to (KW)₄ enhanced the peptides’ antibacterial activity; (KW)₅ exhibited somewhat less bactericidal activity than (KW)₄. Cytotoxicity, measured as lysis of human red blood cells, also increased with increasing chain length. With (KW)₅, reduced antibacterial activity and increased cytotoxicity correlated with greater hydrophobicity and self-aggregation in the aqueous environment. The peptides acted by inducing rapid collapse of the bacterial transmembrane potential and induction of membrane permeability. The mode of interaction of the peptides and the phosphate groups of lipopolysaccharide was dependent upon the peptides’ ability to permeate the membrane. Longer peptides [(KW)₄ and (KW)₅] but not shorter peptides [(KW)₂ and (KW)₃] strongly bound and partially inserted into negatively charged, anionic lipid bilayers. These longer peptides also induced membrane permeabilization and aggregation of lipid vesicles. The peptides had a disordered structure in aqueous solution, and only (KW)₄ and (KW)₅ displayed a folded conformation on lipid membranes. Moreover, (KW)₄ destroyed and agglutinated bacterial cells, demonstrating its potential as an antimicrobial agent. Collectively, the results show (KW)₄ to be the most efficacious peptide in the (KW) n series, exhibiting strong antibacterial activity with little cytotoxicity.

Keywords: Lysine; Tryptophan; Amidation; Antimicrobial peptides; Membrane permeabilization; Bacterial agglutination

Second-tier test for quantification of underivatized amino acids in dry blood spot for metabolic diseases in newborn screening by Chunyan Wang; Hongbin Zhu; Wenyan Zhang; Fengrui Song; Zhiqiang Liu; Shuying Liu (pp. 661-671).

The quantitative analysis of amino acids (AAs) in single dry blood spot (DBS) samples is an important issue for metabolic diseases as a second-tier test in newborn screening. An analytical method for quantifying underivatized AAs in DBS was developed by using liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS). The sample preparation in this method is simple and ion-pairing agent is not used in the mobile phase that could avoid ion suppression, which happens in mass spectrometry and avoids damage to the column. Through chromatographic separation, some isomeric compounds could be identified and quantified, which cannot be solved through only appropriate multiple reactions monitoring transitions by MS/MS. The concentrations of the different AAs were determined using non-deuterated internal standard. All calibration curves showed excellent linearity within test ranges. For most of the amino acids the accuracy of extraction recovery was between 85.3 and 115 %, and the precision of relative standard deviation was <7.0 %. The 35 AAs could be identified in DBS specimens by the developed LC–MS/MS method in 17–19 min, and eventually 24 AAs in DBS were quantified. The results of the present study prove that this method as a second-tier test in newborn screening for metabolic diseases could be performed by the quantification of free AAs in DBS using the LC–MS/MS method. The assay has advantages of high sensitive, specific, and inexpensive merits because non-deuterated internal standard and acetic acid instead of ion-pairing agent in mobile phase are used in this protocol.

Keywords: Newborn screening; Dry blood spot; Underivatized amino acids; LC–MS/MS

A novel radiofluorinated agouti-related protein for tumor angiogenesis imaging by Han Jiang; Sarah J. Moore; Shuanglong Liu; Hongguang Liu; Zheng Miao; Frank V. Cochran; Yang Liu; Mei Tian; Jennifer R. Cochran; Hong Zhang; Zhen Cheng (pp. 673-681).

A novel protein scaffold based on the cystine knot domain of the agouti-related protein (AgRP) has been used to engineer mutants that can bind to the α_vβ₃ integrin receptor with high affinity and specificity. In the current study, an ¹⁸F-labeled AgRP mutant (7C) was prepared and evaluated as a positron emission tomography (PET) probe for imaging tumor angiogenesis. AgRP-7C was synthesized by solid phase peptide synthesis and site-specifically conjugated with 4-nitrophenyl 2-^18/19F-fluoropropionate (^18/19F-NFP) to produce the fluorinated peptide, ^18/19F-FP-AgRP-7C. Competition binding assays were used to measure the relative affinities of AgRP-7C and ¹⁹F-FP-AgRP-7C to human glioblastoma U87MG cells that overexpress α_vβ₃ integrin. In addition, biodistribution, metabolic stability, and small animal PET imaging studies were conducted with ¹⁸F-FP-AgRP-7C using U87MG tumor-bearing mice. Both AgRP-7C and ¹⁹F-FP-AgRP-7C specifically competed with ¹²⁵I-echistatin for binding to U87MG cells with half maximal inhibitory concentration (IC₅₀) values of 9.40 and 8.37 nM, respectively. Non-invasive small animal PET imaging revealed that ¹⁸F-FP-AgRP-7C exhibited rapid and good tumor uptake (3.24 percentage injected dose per gram [% ID/g] at 0.5 h post injection [p.i.]). The probe was rapidly cleared from the blood and from most organs, resulting in excellent tumor-to-normal tissue contrasts. Tumor uptake and rapid clearance were further confirmed with biodistribution studies. Furthermore, co-injection of ¹⁸F-FP-AgRP-7C with a large molar excess of blocking peptide c(RGDyK) significantly inhibited tumor uptake in U87MG xenograft models, demonstrating the integrin-targeting specificity of the probe. Metabolite assays showed that the probe had high stability, making it suitable for in vivo applications. ¹⁸F-FP-AgRP-7C exhibits promising in vivo properties such as rapid tumor targeting, good tumor uptake, and excellent tumor-to-normal tissue ratios, and warrants further investigation as a novel PET probe for imaging tumor angiogenesis.

Keywords: Knottin; α_vβ₃ integrin; PET; ¹⁸F

On the role of the mitochondrial 2-oxoglutarate dehydrogenase complex in amino acid metabolism by Wagner L. Araújo; Lidia Trofimova; Garik Mkrtchyan; Dirk Steinhauser; Leonard Krall; Anastasia Graf; Alisdair R. Fernie; Victoria I. Bunik (pp. 683-700).

Mitochondria are tightly linked to cellular nutrient sensing, and provide not only energy, but also intermediates for the de novo synthesis of cellular compounds including amino acids. Mitochondrial metabolic enzymes as generators and/or targets of signals are therefore important players in the distribution of intermediates between catabolic and anabolic pathways. The highly regulated 2-oxoglutarate dehydrogenase complex (OGDHC) participates in glucose oxidation via the tricarboxylic acid cycle. It occupies an amphibolic branch point in the cycle, where the energy-producing reaction of the 2-oxoglutarate degradation competes with glutamate (Glu) synthesis via nitrogen incorporation into 2-oxoglutarate. To characterize the specific impact of the OGDHC inhibition on amino acid metabolism in both plant and animal mitochondria, a synthetic analog of 2-oxoglutarate, namely succinyl phosphonate (SP), was applied to living systems from different kingdoms, both in situ and in vivo. Using a high-throughput mass spectrometry-based approach, we showed that organisms possessing OGDHC respond to SP by significantly changing their amino acid pools. By contrast, cyanobacteria which lack OGDHC do not show perturbations in amino acids following SP treatment. Increases in Glu, 4-aminobutyrate and alanine represent the most universal change accompanying the 2-oxoglutarate accumulation upon OGDHC inhibition. Other amino acids were affected in a species-specific manner, suggesting specific metabolic rearrangements and substrate availability mediating secondary changes. Strong perturbation in the relative abundance of amino acids due to the OGDHC inhibition was accompanied by decreased protein content. Our results provide specific evidence of a considerable role of OGDHC in amino acid metabolism.

Keywords: Amino acid metabolism; Mitochondria; 2-oxoglutarate dehydrogenase; Succinyl phosphonate; TCA cycle

Covalent fusion inhibitors targeting HIV-1 gp41 deep pocket by Yu Bai; Huifang Xue; Kun Wang; Lifeng Cai; Jiayin Qiu; Shuangyu Bi; Luhua Lai; Maosheng Cheng; Shuwen Liu; Keliang Liu (pp. 701-713).

Covalent inhibitors form covalent adducts with their target, thus permanently inhibiting a physiological process. Peptide fusion inhibitors, such as T20 (Fuzeon, enfuvirtide) and C34, interact with the N-terminal heptad repeat of human immunodeficiency virus type 1 (HIV-1) gp41 glycoprotein to form an inactive hetero six-helix bundle (6-HB) to prevent HIV-1 infection of host cells. A covalent strategy was applied to peptide fusion inhibitor design by introducing a thioester group into C34-like peptide. The modified peptide maintains the specific interaction with its target N36. After the 6-HB formation, a covalent bond between C- and N-peptides was formed by an inter-helical acyl transfer reaction, as characterized by various biophysical and biochemical methods. The covalent reaction between the reactive C-peptide fusion inhibitor and its N-peptide target is highly selective, and the reaction greatly increases the thermostability of the 6-HB. The modified peptide maintains high potency against HIV-1-mediated cell–cell fusion and infection.

Keywords: HIV-1; Gp41; Peptide; Six-helix bundle; Covalent inhibitor

Transformation of the naturally occurring frog skin peptide, alyteserin-2a into a potent, non-toxic anti-cancer agent by J. Michael Conlon; Milena Mechkarska; Manju Prajeep; Kholoud Arafat; Milan Zaric; Miodrag L. Lukic; Samir Attoub (pp. 715-723).

Alyteserin-2a (ILGKLLSTAAGLLSNL.NH₂) is a cationic, amphipathic α-helical cell-penetrating peptide, first isolated from skin secretions of the midwife toad Alytes obstetricans. Structure–activity relationships were investigated by synthesizing analogs of alyteserin-2a in which amino acids on the hydrophobic face of the helix were replaced by l-tryptophan and amino acids on the hydrophilic face were replaced by one or more l-lysine or d-lysine residues. The Trp-containing peptides display increased cytotoxic activity against non-small cell lung adenocarcinoma A549 cells (up to 11-fold), but hemolytic activity against human erythrocytes increases in parallel. The potency of the N15K analog against A549 cells (LC₅₀ = 13 μM) increases sixfold relative to alyteserin-2a and the therapeutic index (ratio of LC₅₀ for erythrocytes and tumor cells) increases twofold. Incorporation of a d-Lys¹¹ residue into the N15K analog generates a peptide that retains potency against A549 cells (LC₅₀ = 15 μM) but whose therapeutic index is 13-fold elevated relative to the native peptide. [G11k, N15K] alyteserin-2a is also active against human hepatocarcinoma HepG2 cells (LC₅₀ = 26 μM), breast adenocarcinoma MDA-MB-231 cells (LC₅₀ = 20 μM), and colorectal adenocarcinoma HT-29 cells (LC₅₀ = 28 μM). [G11k, N15K] alyteserin-2a, in concentrations as low as 1 μg/mL, significantly (P < 0.05) inhibits the release of the immune-suppressive cytokines IL-10 and TGF-β from unstimulated and concanavalin A-stimulated peripheral blood mononuclear cells. The data suggest a strategy of increasing the cationicity while reducing the helicity of naturally occurring amphipathic α-helical peptides to generate analogs with improved cytotoxicity against tumor cells but decreased activity against non-neoplastic cells.

Keywords: Cell-penetrating peptide; Alyteserin-2a; Structure–activity; Anticancer

Isoleucine as a possible bridge between exogenous delivery and terrestrial enhancement of homochirality by Feng Li; Daniel Fitz; Bernd M. Rode (pp. 725-732).

We report a highly enantioselective oligomerization of isoleucine stereomers in the salt-induced peptide formation reaction under plausibly prebiotic earth conditions. Up to 6.5-fold superiority in reactivity of l-isoleucine was observed, compared to its d-enantiomer, after 14 evaporation cycles in the presence of Cu²⁺ and NaCl. Since isoleucine is among the proteinogenic amino acids that were found enantioenriched in meteorites, this present work may further correlate the extraterrestrial delivery and endogenous production of biological homochirality by virtue of a protein constituent rather than the rarely occurring α-methylated amino acids.

Keywords: Isoleucine; Allo-isoleucine; Homochirality; Salt-induced pepetide formation

Supported oligomethionine sulfoxide and Ellman’s reagent for cysteine bridges formation by Luisa Ronga; Pascal Verdié; Pierre Sanchez; Christine Enjabal; Amélie Maurras; Magalie Jullian; Karine Puget; Jean Martinez; Gilles Subra (pp. 733-742).

A large number of bioactive peptides are cyclized through a disulfide bridge. This structural feature is very important for both bioactivity and stability. The oxidation of cysteine side chains is challenging not only to avoid intermolecular reaction leading to oligomers and oxidation of other residues but also to remove solvents and oxidant such as dimethyl sulfoxide. Supported reagents advantageously simplify the work-up of such disulfide bond formation, but may lead to a significant decrease in yield of the oxidized product. In this study, two resins working through different mechanisms were evaluated: Clear-Ox, a supported version of Ellman’s reagent and Oxyfold, consisting in a series of oxidized methionine residues. The choice of the supported reagent is discussed on the light of reaction speed, side-products formation and yield considerations.

Keywords: Oxidation; Supported reagent; Disulfide bond; Cyclic peptide; Methionine; Trisulfide; Oxyfold; Clear-Ox

Effect of oxygen deficiency on nitrogen assimilation and amino acid metabolism of soybean root segments by Halley Caixeta Oliveira; Ladaslav Sodek (pp. 743-755).

Plants submitted to O₂ deficiency present a series of biochemical modifications, affecting overall root metabolism. Here, the effect of hypoxia on the metabolic fate of ¹⁵N derived from ¹⁵NO₃ ⁻, ¹⁵NO₂ ⁻ and ¹⁵NH₄ ⁺ in isolated soybean root segments was followed by gas chromatography–mass spectrometry, to provide a detailed analysis of nitrogen assimilation and amino acid biosynthesis under hypoxia. O₂ deficiency decreased the uptake of the nitrogen sources from the solution, as ratified by the lower ¹⁵NO₃ ⁻ and ¹⁵NH₄ ⁺ enrichment in the root segments. Moreover, analysis of endogenous NO₂ ⁻ and ¹⁵NH₄ ⁺ levels suggested a slower metabolism of these ions under hypoxia. Accordingly, regardless of the nitrogen source, hypoxia reduced total ¹⁵N incorporation into amino acids. Analysis of ¹⁵N enrichment patterns and amino acid levels suggest a redirecting of amino acid metabolism to alanine and γ-aminobutyric acid synthesis under hypoxia and a differential sensitivity of individual amino acid pathways to this stress. Moreover, the role of glutamine synthetase in nitrogen assimilation both under normoxia and hypoxia was ratified. In comparison with ¹⁵NH₄ ⁺, ¹⁵NO₂ ⁻ assimilation into amino acids was more strongly affected by hypoxia and NO₂ ⁻ accumulated in root segments during this stress, indicating that nitrite reductase may be an additional limiting step. NO₂ ⁻ accumulation was associated with a higher nitric oxide emission. ¹⁵NO₃ ⁻ led to much lower ¹⁵N incorporation in both O₂ conditions, probably due to the limited nitrate reductase activity of the root segments. Overall, the present work shows that profound alterations of root nitrogen metabolism occur during hypoxic stress.

Keywords: Amino acid; Ammonium; Hypoxia; Nitrate; Nitrite; Nitrogen

Glutamate induces neutrophil cell migration by activating class I metabotropic glutamate receptors by Rahul Gupta; Santanu Palchaudhuri; Dhrubajyoti Chattopadhyay (pp. 757-767).

Leukocytes are recruited at the site of infection or injury as a part of the innate immune system, and play a very critical role in fighting the invading microorganisms and/or healing wounds. Neutrophils are the most abundant leukocytes in healthy humans and are the principal cell types that arrive at the target site in the initial phase of this process. Previous studies from our laboratory have shown that the amino acid glutamate is a novel chemotaxis-inducing factor for human neutrophils. In this report, we provide evidences that clearly demonstrate that the glutamate-induced neutrophil cell migration activity is mediated by the class I metabotropic glutamate receptors. Our results further show that a specific integrin β2 (ITG β2) receptor, namely LFA1 (α_Lβ₂) is activated upon glutamate treatment and is required for further downstream signaling events leading to increased migration of human neutrophil cells. Following glutamate stimulation, LFA1 is phosphorylated by the Src Kinase Lck at the Y735 residue, which triggers a downstream signaling cascade leading to activation of PI3K, Syk, Vav and finally the Rho family GTPase, Rac2. Interestingly, glutamate was previously found to be present in elevated levels in wound fluid. Furthermore, glutamate level was also found to go up following inflammation. Taken together, our study suggests a novel mode of neutrophil recruitment to the target site following an infection or injury.

Keywords: Neutrophil recruitment; Chemotaxis; Glutamate receptor; Integrin

Synthesis of 4-hydroxy-β³-homoprolines and their insertion in α/β/α-tripeptides by Franca M. Cordero; Carolina Vurchio; Marco Lumini; Alberto Brandi (pp. 769-780).

The stereoselective syntheses of 2-cyclopropyl- and (2S)-2-hydroxymethyl-(3R,4S)-4-hydroxy-β³-homoproline are described. The reported amino acids were constructed through 1,3-dipolar cycloaddition of strained alkylidenecyclopropanes with enantiopure pyrroline N-oxides derived from malic acid followed by thermal rearrangement of the adducts in the presence of trifluoroacetic acid. The two-step sequence afforded the homoprolines suitably protected to be directly used as building blocks in peptidomimetic synthesis as proved by the synthesis of the two model mixed α/β/α tripeptides Phe-β³-HPro-Val.

Keywords: Cycloaddition; Rearrangement; Peptidomimetics; Heterocycles; Small ring systems

A proteomic Ramachandran plot (PRplot) by Oliviero Carugo; Kristina Djinović-Carugo (pp. 781-790).

Each protein structure can be characterized by the average values of the main chain torsion angles ϕ and ψ and, as a consequence, be plotted on a bidimensional diagram, which resembles the Ramachandran plot. Here, we describe a proteomic ϕ–ψ plot (PRplot) where each protein structure is associated with one point, allowing in this way to represent the entire protein structure universe. It was verified that the PRplot is a robust tool since it does not depend on the dimension of the proteins, on the crystallographic resolution of the structures, nor on the biological source; moreover, it is little affected by disordered and structurally uncharacterized residues. The proteins mapped on the PRplot tend to cluster in three regions that correspond to the structures rich in alpha-helices, in beta-strands, and in both helices and strands, and are distributed along a sigmoidal curve that connect these three highly populated regions. PRplots are a unique instrument to project all protein structures on a single bidimensional plane where the entire structural complexity is reduced to a striking simplicity, with the sigmoid curve clearly delineating the space fraction accessible to a stable protein.

Keywords: Protein backbone conformation; Protein fold; Protein structure; Proteome; Ramachandran plot; Structural bioinformatics

Synthesis of polysubstituted β-amino cyclohexane carboxylic acids via Diels–Alder reaction using Ni(II)-complex stabilized β-alanine derived dienes by Xiao Ding; Hengshuai Wang; Jiang Wang; Sinan Wang; Daizong Lin; Li Lv; Yu Zhou; Xiaomin Luo; Hualiang Jiang; José Luis Aceña; Vadim A. Soloshonok; Hong Liu (pp. 791-796).

This paper describes the design and synthesis of a new class of β-alanine derived dienes stabilized by Ni(II)-complex. Preliminary study of their Diels–Alder cycloaddition reactions with several types of dienophiles demonstrates their significant synthetic potential for the preparation of various polyfunctional β-aminocyclohexane carboxylic acids.

Keywords: Diels–Alder reaction; Nickel (II); β-Alanine; β-Aminocyclohexane carboxylic acids

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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.44, #2)

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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.44, #2)