Amino Acids (v.46, #4)

Positioning of aminopeptidase inhibitors in next generation cancer therapy by Sarina M. Hitzerd; Sue Ellen Verbrugge; Gert Ossenkoppele; Gerrit Jansen; Godefridus J. Peters (793-808).
Aminopeptidases represent a class of (zinc) metalloenzymes that catalyze the cleavage of amino acids nearby the N-terminus of polypeptides, resulting in hydrolysis of peptide bonds. Aminopeptidases operate downstream of the ubiquitin–proteasome pathway and are implicated in the final step of intracellular protein degradation either by trimming proteasome-generated peptides for antigen presentation or full hydrolysis into free amino acids for recycling in renewed protein synthesis. This review focuses on the function and subcellular location of five key aminopeptidases (aminopeptidase N, leucine aminopeptidase, puromycin-sensitive aminopeptidase, leukotriene A4 hydrolase and endoplasmic reticulum aminopeptidase 1/2) and their association with different diseases, in particular cancer and their current position as target for therapeutic intervention by aminopeptidase inhibitors. Historically, bestatin was the first prototypical aminopeptidase inhibitor that entered the clinic 35 years ago and is still used for the treatment of lung cancer. More recently, new generation aminopeptidase inhibitors became available, including the aminopeptidase inhibitor prodrug tosedostat, which is currently tested in phase II clinical trials for acute myeloid leukemia. Beyond bestatin and tosedostat, medicinal chemistry has emerged with additional series of potential aminopeptidases inhibitors which are still in an early phase of (pre)clinical investigations. The expanded knowledge of the unique mechanism of action of aminopeptidases has revived interest in aminopeptidase inhibitors for drug combination regimens in anti-cancer treatment. In this context, this review will discuss relevant features and mechanisms of action of aminopeptidases and will also elaborate on factors contributing to aminopeptidase inhibitor efficacy and/or loss of efficacy due to drug resistance-related phenomena. Together, a growing body of data point to aminopeptidase inhibitors as attractive tools for combination chemotherapy, hence their implementation may be a step forward in a new era of personalized treatment of cancer patients.
Keywords: Amino acids; Aminopeptidases; Aminopeptidase inhibitors; Bestatin; Tosedostat

Plant amino acid-derived vitamins: biosynthesis and function by Javier A. Miret; Sergi Munné-Bosch (809-824).
Vitamins are essential organic compounds for humans, having lost the ability to de novo synthesize them. Hence, they represent dietary requirements, which are covered by plants as the main dietary source of most vitamins (through food or livestock’s feed). Most vitamins synthesized by plants present amino acids as precursors (B1, B2, B3, B5, B7, B9 and E) and are therefore linked to plant nitrogen metabolism. Amino acids play different roles in their biosynthesis and metabolism, either incorporated into the backbone of the vitamin or as amino, sulfur or one-carbon group donors. There is a high natural variation in vitamin contents in crops and its exploitation through breeding, metabolic engineering and agronomic practices can enhance their nutritional quality. While the underlying biochemical roles of vitamins as cosubstrates or cofactors are usually common for most eukaryotes, the impact of vitamins B and E in metabolism and physiology can be quite different on plants and animals. Here, we first aim at giving an overview of the biosynthesis of amino acid-derived vitamins in plants, with a particular focus on how this knowledge can be exploited to increase vitamin contents in crops. Second, we will focus on the functions of these vitamins in both plants and animals (and humans in particular), to unravel common and specific roles for vitamins in evolutionary distant organisms, in which these amino acid-derived vitamins play, however, an essential role.
Keywords: Thiamine; Riboflavin; Nicotinic acid; Pantothenate; Pyridoxal; Biotin; Folic acid; Tocopherol

Cyclodidepsipeptides with a promising scaffold in medicinal chemistry by Andrija Smelcerovic; Predrag Dzodic; Voja Pavlovic; Emiliya Cherneva; Denitsa Yancheva (825-840).
Among the large family of cyclodepsipeptides, the simplest members are the cyclodidepsipeptides which have an ester group and an amide group in the same six-membered ring. To point out the pharmacological potential of this class of compounds, the present article reviews structure, isolation, synthesis and biological properties of the known cyclodidepsipeptides. Synthesis of cyclodidepsipeptides is achieved by two general approaches—by initial formation of the amide bond, or initial formation of the ester bond; and subsequent intermolecular cyclization to cyclodidepsipeptide structure. It is closely related to the condensation and ring-closure strategies applied in the preparation of the larger members of the cyclodepsipeptide family. However, due to synthesis of the smaller heretocycles it allows for the use of more versatile building blocks. There are data on antimicrobial, antioxidant and immunomodulatory activities of cyclodidepsipeptides as well as their inhibitory activities toward α-glucosidase, acyl-CoA:cholesterol acyltransferase, xanthine oxidase and platelet aggregation. Because we have recently found that two 6-(propan-2-yl)-4-methyl-morpholine-2,5-diones, as novel non-purine xanthine oxidase inhibitors, may give promise to be used in the treatment of gout, in this review we have included a study of molecular interactions of the selected cyclodidepsipeptides with xanthine oxidase using idTarget web server. Cyclodidepsipeptides showed promising pharmacological activities and meet all criteria for good solubility and permeability. However, further research of their medical application is necessary. In addition to this, the diversity of natural cyclodidepsipeptides, simplicity for synthesis and convenience for rational drug design indicate the cyclodidepsipeptide as promising scaffold in medicinal chemistry.
Keywords: Biological activities; Cyclodidepsipeptides; Docking; Drug-likeness; Structure–activity relationships; Synthesis

The 14-3-3 proteins regulate diverse biological processes that are implicated in cancer development, and seven 14-3-3 isoforms were identified with isoform-specific roles in different human tumors. In our previous work, we dissected the interactome of 14-3-3ε formed during the DNA damage response in a hepatocellular carcinoma (HCC) cell using an AACT/SILAC-based quantitative proteomic approach. In this study, we used a similar proteomic approach to profile/identify the 14-3-3ε interactome formed in native HCC cells. Functional categorization and data-dependent network analysis of the native HCC-specific 14-3-3ε interactome revealed that 14-3-3ε is involved in the regulation of multiple biological processes (BPs)/pathways, including cell cycle control, apoptosis, signal transduction, transport, cell adhesion, carbohydrate metabolism, and nucleic acid metabolism. Biological validation further supports that 14-3-3ε, via association with multiple BP/pathway-specific proteins, coordinates the regulation of proliferation, survival, and metastasis of HCC. The findings in this study, together with those of our previous study, provide an extensive profile of the 14-3-3ε interaction network in HCC cells, which should be valuable for understanding the pathology of HCC and HCC therapy.
Keywords: Hepatocellular carcinoma (HCC); 14-3-3ε; AACT/SILAC; LC–MS/MS

Post-translational modification of protein lysines was recently shown to be a common feature of eukaryotic organisms. The ubiquitin modification is regarded as a versatile regulatory mechanism with many important cellular roles. Large-scale datasets are becoming available for H. sapiens ubiquitination. However, using current experimental techniques the vast majority of their sites remain unidentified and in silico tools may offer an alternative. Here, we introduce Rapid UBIquitination (RUBI) a sequence-based ubiquitination predictor designed for rapid application on a genome scale. RUBI was constructed using an iterative approach. At each iteration, important factors which influenced performance and its usability were investigated. The final RUBI model has an AUC of 0.868 on a large cross-validation set and is shown to outperform other available methods on independent sets. Predicted intrinsic disorder is shown to be weakly anti-correlated to ubiquitination for the H. sapiens dataset and improves performance slightly. RUBI predicts the number of ubiquitination sites correctly within three sites for ca. 80 % of the tested proteins. The average potentially ubiquitinated proteome fraction is predicted to be at least 25 % across a variety of model organisms, including several thousand possible H. sapiens proteins awaiting experimental characterization. RUBI can accurately predict ubiquitination on unseen examples and has a signal across different eukaryotic organisms. The factors which influenced the construction of RUBI could also be tested in other post-translational modification predictors. One of the more interesting factors is the influence of intrinsic protein disorder on ubiquitinated lysines where residues with low disorder probability are preferred.
Keywords: Ubiquitination; Ubiquitylation; Lysine; Machine learning; Intrinsic disorder

The endogenous sulfur-containing amino acid l-cysteine injected into the cerebrospinal fluid space of the cisterna magna increases arterial blood pressure (ABP) and heart rate (HR) in the freely moving rat. The present study examined (1) cardiovascular responses to l-cysteine microinjected into the rostral ventrolateral medulla (RVLM), where a group of neurons regulate activities of cardiovascular sympathetic neurons and (2) involvement of ionotropic excitatory amino acid (iEAA) receptors in response. In the RVLM of urethane-anesthetized rats accessed ventrally and identified with pressor responses to l-glutamate (10 mM, 34 nl), microinjections of l-cysteine increased ABP and HR dose dependently (3–100 mM, 34 nl). The cardiovascular responses to l-cysteine (30 mM) were not attenuated by a prior injection of either antagonist alone, MK801 (20 mM, 68 nl) for the NMDA type of iEAA receptors, or CNQX (2 mM) for the non-NMDA type. However, inhibition of both NMDA and non-NMDA receptors with additional prior injection of either antagonist completely blocked those responses to l-cysteine. The results indicate that l-cysteine has functional cardiovascular action in the RVLM of the anesthetized rat, and the responses to l-cysteine involve both NMDA and non-NMDA receptors albeit in a mutually exclusive parallel fashion. The findings may suggest endogenous roles of l-cysteine indirectly via iEAA receptors in the neuronal network of the RVLM for cardiovascular regulation in physiological and pathological situations.
Keywords: l-Cysteine; Arterial blood pressure; Heart rate; RVLM; Rats; Ionotropic excitatory amino acid receptors

The designer leptin antagonist peptide Allo-aca compensates for short serum half-life with very tight binding to the receptor by Laszlo Otvos Jr.; Stefan W. Vetter; Mohit Koladia; Daniel Knappe; Rico Schmidt; Eszter Ostorhazi; Ilona Kovalszky; Nina Bionda; Predrag Cudic; Eva Surmacz; John D. Wade; Ralf Hoffmann (873-882).
The leptin receptor antagonist peptide Allo-aca exhibits picomolar activities in various cellular systems and sub-mg/kg subcutaneous efficacies in animal models making it a prime drug candidate and target validation tool. Here we identified the biochemical basis for its remarkable in vivo activity. Allo-aca decomposed within 30 min in pooled human serum and was undetectable beyond the same time period from mouse plasma during pharmacokinetic measurements. The C max of 8.9 μg/mL at 5 min corresponds to approximately 22 % injected peptide present in the circulation. The half-life was extended to over 2 h in bovine vitreous fluid and 10 h in human tears suggesting potential efficacy in ophthalmic diseases. The peptide retained picomolar anti-proliferation activity against a chronic myeloid leukemia cell line; addition of a C-terminal biotin label increased the IC50 value by approximately 200-fold. In surface plasmon resonance assays with the biotin-labeled peptide immobilized to a NeutrAvidin-coated chip, Allo-aca exhibited exceptionally tight binding to the binding domain of the human leptin receptor with k a = 5 × 105 M−1 s−1 and k diss = 1.5 × 10−4 s−1 values. Peptides excel in terms of high activity and selectivity to their targets, and may activate or inactivate receptor functions considerably longer than molecular turnovers that take place in experimental animals.
Keywords: Anti-proliferation; Dissociation constant; Metabolic stability; Peptide therapeutics; Receptor activation

Dietary arginine supplementation enhances intestinal expression of SLC7A7 and SLC7A1 and ameliorates growth depression in mycotoxin-challenged pigs by Jie Yin; Wenkai Ren; Jielin Duan; Li Wu; Shuai Chen; Tiejun Li; Yulong Yin; Guoyao Wu (883-892).
This study tested the hypothesis that dietary l-arginine supplementation confers beneficial effects on growing pigs fed a mold-contaminated diet. The measured variables included: (1) the average daily weight gain and feed:gain ratio; (2) activities of total superoxide dismutase, glutathione peroxidase, diamine oxidase, as well as amino acid and d-lactate concentrations in serum; (3) intestinal morphology; (4) expression of the genes for SLC7A7 (amino acid transporter light chain, y+L system, family 7, member 7), SLC7A1 (cationic amino acid transporter, y+ system, family 7, member 1), SLC1A1 (neuronal/epithelial high affinity glutamate transporter, system XAG, member 1), SLC5A1 (sodium/glucose cotransporter, family 5, member 1) in the ileum and jejunum. Mycotoxins in feedstuffs resulted in an enlarged small intestine mass, oxidative injury in tissues, and reduced growth performance in pigs. Dietary arginine supplementation enhanced (P < 0.05) expression of jejunal SLC7A7 and ileal SLC7A1, in comparison with the control and mycotoxin groups. In addition, supplementing 1 % l-arginine to the mycotoxin-contaminated feed had the following beneficial effects (P < 0.05): (1) alleviating the imbalance of the antioxidant system in the body; (2) ameliorating intestinal abnormalities; and (3) attenuating whole-body growth depression, compared with the mycotoxin group without arginine treatment. Collectively, these results indicate that dietary supplementation with l-arginine exerts a protective role in pigs fed mold-contaminated foods. The findings may have important nutritional implications for humans and other mammals.
Keywords: Arginine; Mycotoxin; Small intestine; Swine; Nutrition

Homocysteinylation score of high-molecular weight plasma proteins by Alexandr A. Zhloba; Tatiana F. Subbotina (893-899).
Ultrafiltration rates (filterability) of protein-bound homocysteine (Hcy), unlike glutathione (Glt), are significantly decreased in patients with cardiovascular disorders, end-stage renal disease, and prothrombotic conditions. Reduced filterability of Hcy through 300,000 MWCO PES membranes in these groups of patients was observed, regardless of hyperhomocysteinemia (HHcy) degree. Filterability of Hcy, but not of glutathione, was impaired in plasma preparations. It is possible that the patients with impaired filterability of Hcy in mixed disulfide state are characterized by increased ability of partial Hcy retention by proteins and complexes with higher than albumin molecular weight. These findings led us to conclude that evaluation of protein-bound Hcy filterability may provide more complete diagnostic data, to interpret clinical significance of HHcy. The proposed Hcy filterability test may be performed as a simple laboratory procedure, in addition to conventional tests for total Hcy.
Keywords: Homocysteine; Glutathione; Centrifugal ultrafiltration

Hylaranins: prototypes of a new class of amphibian antimicrobial peptide from the skin secretion of the oriental broad-folded frog, Hylarana latouchii by Yan Lin; Nan Hu; Peng Lyu; Jie Ma; Lei Wang; Mei Zhou; Suhua Guo; Tianbao Chen; Chris Shaw (901-909).
Amphibian skin secretions contain a broad spectrum of biologically active compounds, particularly antimicrobial peptides, which are considered to constitute a first line of defence against bacterial infection. Here we describe the identification of two prototype peptides representing a novel structural class of antimicrobial peptide from the skin secretion of the oriental broad-folded frog, Hylarana latouchii. Named hylaranin-L1 (GVLSAFKNALPGIMKIIVamide) and hylaranin-L2 (GVLSVIKNALPGIMRFIAamide), both peptides consist of 18 amino acid residues, are C-terminally amidated and are of unique primary structures. Their primary structures were initially deduced by MS/MS fragmentation sequencing from reverse-phase HPLC fractions of skin secretion that demonstrated antimicrobial activity. Subsequently, their precursor-encoding cDNAs were cloned from a skin secretion-derived cDNA library and their primary structures were confirmed unequivocally. Synthetic replicates of both peptides exhibited broad-spectrum antimicrobial activity with mean inhibitory concentrations (MICs) of 34 μM against Gram-negative Escherichia coli, 4.3 μM against Gram-positive Staphylococcus aureus and 4–9 μM against the yeast, Candida albicans. Both peptides exhibited little haemolytic activity (<6 %) at the MICs for S. aureus and C. albicans. Amphibian skin secretions thus continue to provide novel antimicrobial peptide structures that may prove to be lead compounds in the design of new classes of anti-infection therapeutics.
Keywords: Amphibian; Antimicrobial; Peptide; Molecular cloning; Mass spectrometry

Comparison of amino acids interaction with gold nanoparticle by Fatemeh Ramezani; Massoud Amanlou; Hashem Rafii-Tabar (911-920).
The study of nanomaterial/biomolecule interface is an important emerging field in bionanoscience, and additionally in many biological processes such as hard-tissue growth and cell-surface adhesion. To have a deeper understanding of the amino acids/gold nanoparticle assemblies, the adsorption of these amino acids on the gold nanoparticles (GNPs) has been investigated via molecular dynamics simulation. In these simulations, all the constituent atoms of the nanoparticles were considered to be dynamic. The geometries of amino acids, when adsorbed on the nanoparticle, were studied and their flexibilities were compared with one another. The interaction of each of 20 amino acids was considered with 3 and 8 nm gold GNPs.
Keywords: Gold nanoparticle; Amino acid; Molecular dynamic; OPLSAA

The zebrafish is increasingly utilized in the analysis of the effects of ethanol (alcohol) on brain function and behavior. We have shown significant population-dependent alcohol-induced changes in zebrafish behavior and have started to analyze alterations in dopaminergic and serotoninergic responses. Here, we analyze the effects of alcohol on levels of selected neurochemicals using a 2 × 3 (chronic × acute) between-subject alcohol exposure paradigm randomized for two zebrafish populations, AB and SF. Each fish first received the particular chronic treatment (0 or 0.5 vol/vol % alcohol) and subsequently the acute exposure (0, 0.5 or 1.0 % alcohol). We report changes in levels of dopamine, DOPAC, serotonin, 5HIAA, glutamate, GABA, aspartate, glycine and taurine as quantified from whole brain extracts using HPLC. We also analyze monoamine oxidase and tyrosine hydroxylase enzymatic activity. The results demonstrate that compared to SF, AB is more responsive to both acute alcohol exposure and acute alcohol withdrawal at the level of neurochemistry, a finding that correlates well with prior behavioral observations and one which suggests the involvement of genes in the observed alcohol effects. We discuss correlations between the current results and prior behavioral findings, and stress the importance of characterization of zebrafish strains for future behavior genetic and psychopharmacology studies.
Keywords: Acute and chronic alcohol; Ethanol; Ethyl alcohol; Zebrafish; Neurochemistry

Unnatural amino acids increase activity and specificity of synthetic substrates for human and malarial cathepsin C by Marcin Poreba; Marko Mihelic; Priscilla Krai; Jelena Rajkovic; Artur Krezel; Malgorzata Pawelczak; Michael Klemba; Dusan Turk; Boris Turk; Rafal Latajka; Marcin Drag (931-943).
Mammalian cathepsin C is primarily responsible for the removal of N-terminal dipeptides and activation of several serine proteases in inflammatory or immune cells, while its malarial parasite ortholog dipeptidyl aminopeptidase 1 plays a crucial role in catabolizing the hemoglobin of its host erythrocyte. In this report, we describe the systematic substrate specificity analysis of three cathepsin C orthologs from Homo sapiens (human), Bos taurus (bovine) and Plasmodium falciparum (malaria parasite). Here, we present a new approach with a tailored fluorogenic substrate library designed and synthesized to probe the S1 and S2 pocket preferences of these enzymes with both natural and a broad range of unnatural amino acids. Our approach identified very efficiently hydrolyzed substrates containing unnatural amino acids, which resulted in the design of significantly better substrates than those previously known. Additionally, in this study significant differences in terms of the structures of optimal substrates for human and malarial orthologs are important from the therapeutic point of view. These data can be also used for the design of specific inhibitors or activity-based probes.
Keywords: Cysteine protease; Non-proteinogenic; Unnatural amino acid; Substrate library; Fluorogenic substrate

Inexpensive chemical method for preparation of enantiomerically pure phenylalanine by Hiroki Moriwaki; Daniel Resch; Hengguang Li; Iwao Ojima; Ryosuke Takeda; José Luis Aceña; Vadim Soloshonok (945-952).
Here, we report the most inexpensive procedure for chemical synthesis of enantiomerically pure phenylalanine. As a source of chirality, we use the ultimately inexpensive chiral auxiliary, 1-(phenyl)ethylamine, incorporated into the specially designed ligands which form the corresponding intermediate Ni(II) complexes with racemic phenylalanine. Diastereomerically pure Ni(II) complexes, containing either (S)- or (R)-phenylalanine, were disassembled to produce enantiomerically pure target amino acid, along with recycling the chiral ligand. All reactions were conducted under operationally convenient conditions, featuring high yields and thus underscoring attractive cost structure of this method.
Keywords: Phenylalanine; Schiff bases; Ni(II) complexes; Stereogenic nitrogen; Asymmetric synthesis

Disturbance of Arabidopsis thaliana microRNA-regulated pathways by Xcc bacterial effector proteins by Nilubon Kurubanjerdjit; Jeffrey J. P. Tsai; Chien-Hung Huang; Ka-Lok Ng (953-961).
Plants are continuously subjected to infection by pathogens, including bacteria and viruses. Bacteria can inject a variety of effector proteins into the host to reprogram host defense mechanism. It is known that microRNAs participate in plant disease resistance to bacterial pathogens and previous studies have suggested that some bacterial effectors have evolved to disturb the host’s microRNA-regulated pathways; and so enabling infection. In this study, the inter-species interaction between an Xanthomonas campestris pv campestris (Xcc) pathogen effector and Arabidopsis thaliana microRNA transcription promoter was investigated using three methods: (1) interolog, (2) alignment based on using transcription factor binding site profile matrix, and (3) the web-based binding site prediction tool, PATSER. Furthermore, we integrated another two data sets from our previous study into the present web-based system. These are (1) microRNA target genes and their downstream effects mediated by protein–protein interaction (PPI), and (2) the XccArabidopsis PPI information. This present work is probably the first comprehensive study of constructing pathways that comprises effector, microRNA, target genes and PPI for the study of pathogen–host interactions. It is expected that this study may help to elucidate the role of pathogen–host interplay in a plant’s immune system. The database is freely accessible at: .
Keywords: Arabidopsis thaliana ; Xanthomonas campestris pv campestris; Bacterial effector protein; Protein–protein interaction; Pathogen–host interactions; Transcription factor binding site

Alterations in the metabolomics of sulfur-containing substances in rat kidney by betaine by Young Chul Kim; Do Young Kwon; Ji Hyun Kim (963-968).
Earlier studies have shown that betaine administration may modulate the metabolism of sulfur amino acids in the liver. In this study, we determined the changes in the metabolomics of sulfur-containing substances induced by betaine in the kidney, the other major organ actively involved in the transsulfuration reactions. Male rats received betaine (1 %) in drinking water for 2 weeks before killing. Betaine intake did not affect betaine–homocysteine methyltransferase activity or its protein expression in the renal tissue. Expression of methionine synthase was also unchanged. However, methionine levels were increased significantly both in plasma and kidney. Renal methionine adenosyltransferase activity and S-adenosylmethionine concentrations were increased, but there were no changes in S-adenosylhomocysteine, homocysteine, cysteine levels or cystathionine β-synthase expression. γ-Glutamylcysteine synthetase expression or glutathione levels were not altered, but cysteine dioxygenase and taurine levels were decreased significantly. In contrast, betaine administration induced cysteine sulfinate decarboxylase and its metabolic product, hypotaurine. These results indicate that the metabolomics of sulfur-containing substances in the kidney is altered extensively by betaine, although the renal capacity for methionine synthesis is unresponsive to this substance unlike that of the liver. It is suggested that the increased methionine availability due to an enhancement of its uptake from plasma may account for the alterations in the metabolomics of sulfur-containing substances in the kidney. Further studies need to be conducted to clarify the physiological/pharmacological significance of these findings.
Keywords: Betaine; Methionine; Kidney; Sulfur amino acid metabolism; S-adenosylmethionine

Glycine inhibits angiogenic signaling in human hepatocellular carcinoma cells by Helge Bruns; Marius Petrulionis; Daniel Schultze; Mohammed Al Saeedi; Shibo Lin; Kenya Yamanaka; Marius Ambrazevičius; Kęstutis Strupas; Peter Schemmer (969-976).
Hepatocellular carcinoma (HCC) is a highly vascularized tumor with limited susceptibility to chemotherapy. Modern targeted therapies are aimed at specific properties of this neoplasm. Glycine is a simple non-essential amino acid with potential antiangiogenic effects. In this study, the amino acid’s effect on angiogenic signaling in an in vitro model of HCC was evaluated. HepG2 and Huh7 cells were treated with glycine-free DMEM supplemented with 0, 0.01, 0.1, 1.0, 2.0, 5.0 and 10 mM glycine. The direct effects of glycine on the viability of HCC cells were monitored using MTT assay. To detect angiogenic signaling, mRNA and protein levels of vascular endothelial growth factor (VEGF-A) were measured using RT-PCR and Western Blot assays. To determine whether or not glycine receptors (GlyR) played a significant role, the specific antagonist, strychnine, was used as a direct inhibitor. Western Blotting was performed to show the presence of GlyR. While there was no direct pro- or antiproliferative effect of either glycine or strychnine in both cell lines, glycine was shown to significantly decrease VEGF-A expression on mRNA and protein level up to 63 % in both cell lines. This effect was blunted by the presence of strychnine. GlyR was also identified in both cell lines. Glycine decreases GlyR-dependent, VEGF-A-mediated, angiogenic signaling in human HCC and thus might be a promising additive to chemotherapy treatment strategies for highly vascularized tumors.
Keywords: Glycine; Hepatocellular carcinoma; Angiogenesis

Development of an accurate and reliable intelligent decision-making method for the construction of cancer diagnosis system is one of the fast growing research areas of health sciences. Such decision-making system can provide adequate information for cancer diagnosis and drug discovery. Descriptors derived from physicochemical properties of protein sequences are very useful for classifying cancerous proteins. Recently, several interesting research studies have been reported on breast cancer classification. To this end, we propose the exploitation of the physicochemical properties of amino acids in protein primary sequences such as hydrophobicity (Hd) and hydrophilicity (Hb) for breast cancer classification. Hd and Hb properties of amino acids, in recent literature, are reported to be quite effective in characterizing the constituent amino acids and are used to study protein foldings, interactions, structures, and sequence-order effects. Especially, using these physicochemical properties, we observed that proline, serine, tyrosine, cysteine, arginine, and asparagine amino acids offer high discrimination between cancerous and healthy proteins. In addition, unlike traditional ensemble classification approaches, the proposed ‘IDM-PhyChm-Ens’ method was developed by combining the decision spaces of a specific classifier trained on different feature spaces. The different feature spaces used were amino acid composition, split amino acid composition, and pseudo amino acid composition. Consequently, we have exploited different feature spaces using Hd and Hb properties of amino acids to develop an accurate method for classification of cancerous protein sequences. We developed ensemble classifiers using diverse learning algorithms such as random forest (RF), support vector machines (SVM), and K-nearest neighbor (KNN) trained on different feature spaces. We observed that ensemble-RF, in case of cancer classification, performed better than ensemble-SVM and ensemble-KNN. Our analysis demonstrates that ensemble-RF, ensemble-SVM and ensemble-KNN are more effective than their individual counterparts. The proposed ‘IDM-PhyChm-Ens’ method has shown improved performance compared to existing techniques.
Keywords: Cancer classification; Ensemble classifier; Random forest; Breast cancer; Protein primary sequences; Amino acid; Physicochemical properties; Hydrophobicity and hydrophilicity

Proteomic differences between white and brown adipocytes by Wei-Qiang Chen; Lin Li; Gert Lubec (995-1008).
Although a series of protein levels from several protein pathways have been shown to differ between white (WA) and brown (BA) adipocytes, proteomic work on this subject with the exception of mitochondrial protein differences is limited. It was, therefore, the aim of the study to compare WA with BA soluble protein levels. Proteins were extracted from WA and BA and the soluble fraction was run on two-dimensional gel electrophoresis. Quantification of spot volume was carried out and protein spots, statistically different between groups (P < 0.01), were in-gel digested with trypsin and peptides were identified using nano-LC–ESI–MS/MS in the CID and ETD mode. Differences between selected proteins were evaluated by immunoblotting. A network was generated using the ingenuity pathway analysis. Five proteins, protein DJ-1, dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex, isocitrate dehydrogenase subunit alpha, electron transfer flavoprotein subunit alpha and immunoglobulin-binding protein 1, were increased in BA based on a gel-based proteomic method and differential expression was verified by immunoblotting. These individual proteins were represented by one spot each and sequence coverages were between 28 and 65 %. A network generated based on these results indicated a link to ubiquitination. Differential protein levels between WA and BA allow interpretation of previous work on adipocyte biochemistry and form the basis for future studies with genetic or pharmacological inhibition of these proteins accompanied by work on phenotype and adipocyte function.
Keywords: White adipocyte; Brown adipocyte; LCMS; Ubiquitination

The antineoplastic effect of carnosine is accompanied by induction of PDK4 and can be mimicked by l-histidine by Ulrike Letzien; Henry Oppermann; Jürgen Meixensberger; Frank Gaunitz (1009-1019).
Carnosine (β-alanyl-l-histidine) is a naturally occurring dipeptide that shows antineoplastic effects in cell culture as well as in animal experiments. Since its mode of action and the targets at the molecular level have not yet been elucidated, we performed qRT-PCR experiments with RNA isolated from glioblastoma cell lines treated with carnosine, β-alanine, l-alanine, l-histidine and the dipeptide l-alanine-l-histidine. The experiments identified a strong induction of expression of the gene encoding pyruvate dehydrogenase 4 (PDK4) under the influence of carnosine and l-histidine, but not by the other substances employed. In addition, inhibition of cell viability was only detected in cells treated with carnosine and l-histidine, with the latter showing a significantly stronger effect than carnosine. Since the tumor cells expressed the tissue form of carnosinase (CN2) but almost no serum carnosinase (CN1), we conclude that cleavage by CN2 is a prerequisite for the antineoplastic effect of carnosine. In addition, enhanced expression of PDK4 under the influence of carnosine/l-histidine opens a new perspective for the interpretation of the ergogenic potential of dietary β-alanine supplementation and adds a new contribution to a growing body of evidence that single amino acids can regulate key metabolic pathways important in health and disease.
Keywords: Carnosine; l-Histidine; PDK4; Glioblastoma; Cancer

Proteomics of larval hemolymph in Bombyx mori reveals various nutrient-storage and immunity-related proteins by Yan Zhang; Zhaoming Dong; Dandan Wang; Yong Wu; Qianru Song; Peiming Gu; Ping Zhao; Qingyou Xia (1021-1031).
The silkworm, Bombyx mori, is an important economic insect for its production of silk. The larvae of many lepidopteran insects are major agricultural pests and often silkworm is explored as a model organism for other lepidopteran pest species. The hemolymph of caterpillars contains a lot of nutrient and immune components. In this study, we applied liquid chromatography–tandem mass spectrometry to gain a better understanding of the larval hemolymph proteomics in B. mori. We identified 752 proteins in hemolymph collected from day-4 fourth instar and day-7 fifth instar. Nearly half the identified proteins (49 %) were predicted to function as binding proteins and 46 % were predicted to have catalytic activities. Apolipophorins, storage proteins, and 30K proteins constituted the most abundant groups of nutrient-storage proteins. Of them, 30K proteins showed large differences between fourth instar larvae and fifth instar larvae. Besides nutrient-storage proteins, protease inhibitors are also expressed very highly in hemolymph. The analysis also revealed lots of immunity-related proteins, including recognition, signaling, effectors and other proteins, comprising multiple immunity pathways in hemolymph. Our data provide an exhaustive research of nutrient-storage proteins and immunity-related proteins in larval hemolymph, and will pave the way for future physiological and pathological studies of caterpillars.
Keywords: Hemolymph; Bombyx mori ; Apolipophorin; Storage protein; 30K protein; Immune

Design and synthesis of anti-cancer cyclopeptides containing triazole skeleton by Fatemeh Tahoori; Saeed Balalaie; Reza Sheikhnejad; Mahnaz Sadjadi; Parvin Boloori (1033-1046).
We describe the design and synthesis of some hypothetical heptapeptides specifically to overcome the neoplastic activity of ras oncogene and their anti-cancer activities were studied. To improve the anti-cancer activity of the synthesized peptides, their structure modifications were done based on a sequential Ugi/Huisgen 1,3-Dipolar cyclization reaction. The cyclopeptides which contained triazole skeleton showed significant anti-cancer activity against cancer cells with mutated ras oncogene such as A549, PC3 and C26 cells. This study clearly shows the importance of triazole skeleton in biological activity of the peptides. It might be possible to overcome the difficulties involved in making complex peptides by employing this elegant chemistry.
Keywords: Ugi ligation; Ligation of peptides; Anti-cancer activity; Cyclopeptides; Click reaction; Huisgen 1,3-Dipolar reaction

A BODIPY-embedding miltefosine analog linked to cell-penetrating Tat(48-60) peptide favors intracellular delivery and visualization of the antiparasitic drug by Beatriz G. de la Torre; Valentín Hornillos; Juan R. Luque-Ortega; M. A. Abengózar; Francisco Amat-Guerri; A. Ulises Acuña; Luis Rivas; David Andreu (1047-1058).
Therapeutic application of many drugs is often hampered by poor or denied access to intracellular targets. A case in point is miltefosine (MT), an orally active antiparasitic drug, which becomes ineffective when parasites develop dysfunctional uptake systems. We report here the synthesis of a fluorescent BODIPY-embedding MT analogue with appropriate thiol functionalization allowing linkage to the cell-penetrating Tat(48-60) peptide through disulfide or thioether linkages. The resulting constructs are efficiently internalized into the otherwise MT-invulnerable R40 Leishmania strain, resulting in fast parasite killing, and hence successful avoidance of the resistance. In the disulfide-linked conjugate, an additional fluoro tag on the Tat moiety allows to monitor its reductive cleavage within the cytoplasm. Terminally differentiated cells such as peritoneal macrophages, impervious to MT unless infected by Leishmania, can uptake the drug in its Tat-conjugated form. The results afford proof-of-principle for using CPP vectors to avert drug resistance in parasites, and/or for tackling leishmaniasis by modulating macrophage uptake.
Keywords: BODIPY; Cell-penetrating peptide; Miltefosine; Dual fluorescent labeling; Reversion of resistance; Monitoring of intracellular drug delivery

Enteral glutamine infusion modulates ubiquitination of heat shock proteins, Grp-75 and Apg-2, in the human duodenal mucosa by Julien Bertrand; Alexis Goichon; Philippe Chan; Saida Azhar; Stéphane Lecleire; Nathalie Donnadieu; David Vaudry; Anne-Françoise Cailleux; Pierre Déchelotte; Moïse Coëffier (1059-1067).
Glutamine, the most abundant amino acid in the human body, plays several important roles in the intestine. Previous studies showed that glutamine may affect protein expression by regulating ubiquitin–proteasome system. We thus aimed to evaluate the effects of gl utamine on ubiquitinated proteins in human duodenal mucosa. Five healthy male volunteers were included and received during 5 h, on two occasions and in a random order, either an enteral infusion of maltodextrins alone (0.25 g kg−1 h−1, control), mimicking carbohydrate-fed state, or maltodextrins with glutamine (0.117 g kg−1 h−1, glutamine). Endoscopic duodenal biopsies were then taken. Total cellular protein extracts were separated by 2D gel electrophoresis and analyzed by an immunodetection using anti-ubiquitin antibody. Differentially ubiquitinated proteins were then identified by liquid chromatography–electrospray ionization MS/MS. Five proteins were differentially ubiquitinated between control and glutamine conditions. Among these proteins, we identified two chaperone proteins, Grp75 and hsp74. Grp75 was less ubiquitinated after glutamine infusion compared with control. In contrast, hsp74, also called Apg-2, was more ubiquitinated after glutamine. In conclusion, we provide evidence that glutamine may regulate ubiquitination processes of specific proteins, i.e., Grp75 and Apg-2. Grp75 has protective and anti-inflammatory properties, while Apg-2 indirectly regulates stress-induced cell survival and proliferation through interaction with ZO-1. Further studies should confirm these results in stress conditions.
Keywords: Glutamine; Ubiquitination; Proteasome; Intestine; Human; Heat shock protein

Reversible protein phosphorylation is one of the most important post-translational modifications, which regulates various biological cellular processes. Identification of the kinase-specific phosphorylation sites is helpful for understanding the phosphorylation mechanism and regulation processes. Although a number of computational approaches have been developed, currently few studies are concerned about hierarchical structures of kinases, and most of the existing tools use only local sequence information to construct predictive models. In this work, we conduct a systematic and hierarchy-specific investigation of protein phosphorylation site prediction in which protein kinases are clustered into hierarchical structures with four levels including kinase, subfamily, family and group. To enhance phosphorylation site prediction at all hierarchical levels, functional information of proteins, including gene ontology (GO) and protein–protein interaction (PPI), is adopted in addition to primary sequence to construct prediction models based on random forest. Analysis of selected GO and PPI features shows that functional information is critical in determining protein phosphorylation sites for every hierarchical level. Furthermore, the prediction results of Phospho.ELM and additional testing dataset demonstrate that the proposed method remarkably outperforms existing phosphorylation prediction methods at all hierarchical levels. The proposed method is freely available at .
Keywords: Phosphorylation; Hierarchical structure; Functional information; Random forest

Simultaneous electrochemical determination of l-cysteine and l-cysteine disulfide at carbon ionic liquid electrode by Afsaneh Safavi; Raheleh Ahmadi; Farzaneh Aghakhani Mahyari (1079-1085).
A linear sweep voltammetric method is used for direct simultaneous determination of l-cysteine and l-cysteine disulfide (cystine) based on carbon ionic liquid electrode. With carbon ionic liquid electrode as a high performance electrode, two oxidation peaks for l-cysteine (0.62 V) and l-cysteine disulfide (1.3 V) were observed with a significant separation of about 680 mV (vs. Ag/AgCl) in phosphate buffer solution (pH 6.0). The linear ranges were obtained as 1.0–450 and 5.0–700 μM and detection limits were estimated to be 0.298 and 4.258 μM for l-cysteine and l-cysteine disulfide, respectively. This composite electrode was applied for simultaneous determination of l-cysteine and l-cysteine disulfide in two real samples, artificial urine and nutrient broth. Satisfactory results were obtained which clearly indicate the applicability of the proposed electrode for simultaneous determination of these compounds in complex matrices.
Keywords: Carbon ionic liquid electrode; l-Cysteine; l-Cysteine disulfide; Simultaneous determination

Novel anticoagulant therapies target specific clotting factors in blood coagulation cascade. Inhibition of the blood coagulation through Factor VIII–Factor IX interaction represents an attractive approach for the treatment and prevention of diseases caused by thrombosis. Our research efforts are continued by the synthesis and biological evaluation of cyclic, head to tail peptides, analogs of the 558–565 sequence of the A2 subunit of FVIII, aiming at the efficient inhibition of Factor VIIIa–Factor IXa interaction. The analogs were synthesized on solid phase using the acid labile 2-chlorotrityl chloride resin, while their anticoagulant activities were examined in vitro by monitoring activated partial thromboplastin time and the inhibition of Factor VIII activity. The results reveal that these peptides provide bases for the development of new anticoagulant agents.
Keywords: Factor VIII; Anticoagulant activity; Activated partial thromboplastin time; Synthetic cyclic peptides; Peptoid-peptides; FVIII–FIX interaction; Antithrombotic agents

Synthesis of a cyclic isostere of α-methyl homoserine by a stereoselective acylation–alkylation sequence of a chiral γ-lactam by Annafelicia Civitavecchia; Gianluca Martelli; Mario Orena; Samuele Rinaldi (1097-1103).
Starting from a chiral 4-hydroxymethyl pyrrolidin-2-one, an isostere of α-methyl homoserine tethered on a γ-lactam ring was prepared exploiting a stereoselective acylation–methylation sequence, followed by Curtius rearrangement, and structural assignment was confirmed by n.O.e. experiments. By reverting the sequence, the 3-carboxy-3-methyl derivative having the opposite configuration at C-3 was obtained with total stereoselection, but Curtius rearrangement invariably afforded only inseparable mixtures of decomposition products.
Keywords: Conformational constriction; Amino acids; Lactams; Alkylation; Stereoselection

In this paper, we present a pipeline to perform improved QSAR analysis of peptides. The modeling involves a double selection procedure that first performs feature selection and then conducts sample selection before the final regression analysis. Five hundred and thirty-one physicochemical property parameters of amino acids were used as descriptors to characterize the structure of peptides. These high-dimensional descriptors then go through a feature selection process given by the binary matrix shuffling filter (BMSF) to obtain a set of important low-dimensional features. Each descriptor that passes the BMSF filtering also receives a weight defined through its contribution to reduce the estimation error. These selected features served as the predictors for subsequent sample selection and modeling. Based on the weighted Euclidean distances between samples, a common range was determined with high-dimensional semivariogram and then used as a threshold to select the near-neighbor samples from the training set. For each sample to be predicted, the QSAR model was established using SVR with the weighted, selected features based on the exclusive set of near-neighbor training samples. Prediction was conducted for each test sample accordingly. The performances of this pipeline are tested with the QSAR analysis of angiotensin-converting enzyme inhibitors and HLA-A*0201 data sets. Improved prediction accuracy was obtained in both applications. This pipeline can optimize the QSAR modeling from both the feature selection and sample selection perspectives. This leads to improved accuracy over single selection methods. We expect this pipeline to have extensive application prospect in the field of regression prediction.
Keywords: Peptides; Quantitative structure–activity regression; Feature selection; Semivariogram; Support vector regression

The effect of nitrate assimilation deficiency on the carbon and nitrogen status of Arabidopsis thaliana plants by Plínio Rodrigues Santos-Filho; Elzira Elisabeth Saviani; Ione Salgado; Halley Caixeta Oliveira (1121-1129).
Carbon (C) and nitrogen (N) metabolism are integrated processes that modulate many aspects of plant growth, development, and defense. Although plants with deficient N metabolism have been largely used for the elucidation of the complex network that coordinates the C and N status in leaves, studies at the whole-plant level are still lacking. Here, the content of amino acids, organic acids, total soluble sugars, starch, and phenylpropanoids in the leaves, roots, and floral buds of a nitrate reductase (NR) double-deficient mutant of Arabidopsis thaliana (nia1 nia2) were compared to those of wild-type plants. Foliar C and N primary metabolism was affected by NR deficiency, as evidenced by decreased levels of most amino acids and organic acids and total soluble sugars and starch in the nia1 nia2 leaves. However, no difference was detected in the content of the analyzed metabolites in the nia1 nia2 roots and floral buds in comparison to wild type. Similarly, phenylpropanoid metabolism was affected in the nia1 nia2 leaves; however, the high content of flavonol glycosides in the floral buds was not altered in the NR-deficient plants. Altogether, these results suggest that, even under conditions of deficient nitrate assimilation, A. thaliana plants are capable of remobilizing their metabolites from source leaves and maintaining the C–N status in roots and developing flowers.
Keywords: Amino acid; Carbon–nitrogen status; Flavonol glycosides; Nitrate reductase; Source–sink interaction

l-Methionine supplementation maintains the integrity and barrier function of the small-intestinal mucosa in post-weaning piglets by Ying Chen; Defa Li; Zhaolai Dai; Xiangshu Piao; Zhenlong Wu; Bin Wang; Yuhua Zhu; Zhikai Zeng (1131-1142).
This study was conducted to test the hypothesis that different dietary Met levels affect small-intestinal mucosal integrity in post-weaning piglets. Two groups of piglets (n = 6/group) were weaned at 28 days of age and randomly allotted to a basal diet (without extra Met supplementation) or a Met-supplemented diet (with 0.12 % l-Met) for 14 days. The standardized ileal digestible (SID) Met levels were 0.24 and 0.35 %, respectively. At days 7 and 14 of the trial, venous blood samples were obtained from piglets, followed by their euthanasia for tissue collection. Piglets fed the diet supplemented with l-Met had a higher average daily gain during days 7–14 and improved feed efficiency during the entire period. Concentrations of sulfur amino acids (SAA), glutamate acid (Glu), glutamine (Gln), and taurine in the plasma and tissues were higher for the piglets in the Met-supplemented group. Met supplementation increased cysteine (Cys) and glutathione (GSH) concentrations in the plasma and tissues, leading to reductions in plasma Cys/CySS redox potential and tissue GSH/GSSH redox potential. The small-intestinal mucosa of Met-supplemented piglets exhibited improved villus architecture, compared with control piglets. Met supplementation increased transepithelial electrical resistance of the jejunal mucosa. Transport of Met, Gln and Cys across the jejunal mucosa did not differ between control and Met-supplemented piglets. The abundance occludin was higher, whereas the abundance of active caspase-3 was lower, in the jejunum of the Met-supplemented piglets. Collectively, adequate dietary Met is required for optimal protein synthesis and mucosal integrity in the small intestine of post-weaning piglets.
Keywords: l-Methionine; Small intestinal mucosa; Piglets

Improvements to polar 2-D electrophoresis for proteomic applications by Rita Polati; Daniela Cecconi; Ryan Dosselli; Mattia Zaccarin; Renato Millioni (1143-1146).
Recently, we reported a new way of performing 2-DE, called P-dimensional electrophoresis (2-PE). In this approach, the second dimension is achieved in a radial gel which can accommodate up to six 7 cm long IPG strips simultaneously, improving reproducibility and throughput power in respect to 2-DE. Nevertheless, 2-PE was up to now limited to the use of only short strips because of technical difficulties. Here, we describe how to load longer strips (e.g., 18–24 cm) on 2-PE and report some representative images for a qualitative assessment.
Keywords: P-dimensional electrophoresis; Spot resolution; Radial protein separation