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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.39, #1)

Restriction factors of retroviral replication: the example of Tripartite Motif (TRIM) protein 5α and 22 by Anna Kajaste-Rudnitski; Cinzia Pultrone; Flavia Marzetta; Silvia Ghezzi; Tiziana Coradin; Elisa Vicenzi (pp. 1-9).

Viral tropism, replication, and pathogenesis are determined by multiple interactions between the pathogen and the host. In the case of retroviruses, and in particular, the human immunodeficiency virus, the specific interaction of the envelope protein with the host receptors and co-receptors is essential to gain entry in the cells. After entry, the success of retroviruses to complete their life cycle depends on a complex interplay between the virus and host proteins. Indeed, the cell environment is endowed with a number of factors that actively block distinct stage(s) in the microbial life cycle. Among these restriction factors, Tripartite Motif-5α (TRIM5α) has been extensively studied; however, other TRIM family members have been demonstrated to be anti-retroviral effector proteins. This article reviews, in particular, the current knowledge on the anti-retroviral effects of TRIM5α and TRIM22.

Keywords: Retroviral infection; Host restriction factors; TRIM family proteins

Positron emission tomography imaging of prostate cancer by Hao Hong; Yin Zhang; Jiangtao Sun; Weibo Cai (pp. 11-27).

Prostate cancer (PCa) is the second leading cause of cancer death among men in the United States. Positron emission tomography (PET), a non-invasive, sensitive, and quantitative imaging technique, can facilitate personalized management of PCa patients. There are two critical needs for PET imaging of PCa, early detection of primary lesions and accurate imaging of PCa bone metastasis, the predominant cause of death in PCa. Because the most widely used PET tracer in the clinic, ¹⁸F-fluoro-2-deoxy-2-d-glucose (¹⁸F-FDG), does not meet these needs, a wide variety of PET tracers have been developed for PCa imaging that span an enormous size range from small molecules to intact antibodies. In this review, we will first summarize small-molecule-based PET tracers for PCa imaging, which measure certain biological events, such as cell membrane metabolism, fatty acid synthesis, and receptor expression. Next, we will discuss radiolabeled amino acid derivatives (e.g. methionine, leucine, tryptophan, and cysteine analogs), which are primarily based on the increased amino acid transport of PCa cells. Peptide-based tracers for PET imaging of PCa, mostly based on the bombesin peptide and its derivatives which bind to the gastrin-releasing peptide receptor, will then be presented in detail. We will also cover radiolabeled antibodies and antibody fragments (e.g. diabodies and minibodies) for PET imaging of PCa, targeting integrin α_vβ₃, EphA2, the epidermal growth factor receptor, or the prostate stem cell antigen. Lastly, we will identify future directions for the development of novel PET tracers for PCa imaging, which may eventually lead to personalized management of PCa patients.

Keywords: Molecular imaging; Prostate cancer; Positron emission tomography; Peptide; Antibody

Metabolic correlations of glucocorticoids and polyamines in inflammation and apoptosis by G. Bjelaković; I. Stojanović; T. Jevtović Stoimenov; D. Pavlović; G. Kocić; S. Rossi; C. Tabolacci; J. Nikolić; D. Sokolović; Lj. Bjelakovic (pp. 29-43).

Glucocorticoid hormones (GC) are essential in all aspects of human health and disease. Their anti-inflammatory and immunosuppressive properties are reasons for therapeutic application in several diseases. GC suppress immune activation and uncontrolled overproduction and release of cytokines. GC inhibit the release of pro-inflammatory cytokines and stimulate the production of anti-inflammatory cytokines. Investigation of GC’s mechanism of action, suggested that polyamines (PA) may act as mediators or messengers of their effects. Beside glucocorticoids, spermine (Spm) is one of endogenous inhibitors of cytokine production. There are many similarities in the metabolic actions of GC and PA. The major mechanism of GC effects involves the regulation of gene expression. PA are essential for maintaining higher order organization of chromatin in vivo. Spermidine and Spm stabilize chromatin and nuclear enzymes, due to their ability to form complexes with negatively charged groups on DNA, RNA and proteins. Also, there is an increasing body of evidence that GC and PA change the chromatin structure especially through acetylation and deacetylation of histones. GC display potent immunomodulatory activities, including the ability to induce T and B lymphocyte apoptosis, mediated via production of reactive oxygen species (ROS) in the mitochondrial pathway. The by-products of PA catabolic pathways (hydrogen peroxide, amino aldehydes, acrolein) produce ROS, well-known cytotoxic agents involved in programmed cell death (PCD) or apoptosis. This review is an attempt in the better understanding of relation between GC and PA, naturally occurring compounds of all eukaryotic cells, anti-inflammatory and apoptotic agents in physiological and pathological conditions connected to oxidative stress or PCD.

Keywords: Glucocorticoids; Polyamines; Inflammation; Mitochondria; ROS; Apoptosis

Nucleobase-containing peptides: an overview of their characteristic features and applications by Giovanni N. Roviello; Ettore Benedetti; Carlo Pedone; Enrico M. Bucci (pp. 45-57).

Reports on nucleobase-containing chiral peptides (both natural and artificial) and achiral pseudopeptides are reviewed. Their synthesis, structural features, DNA and RNA-binding ability, as well as some other interesting applications which make them promising diagnostic/therapeutic agents of great importance in many areas of biology and therapy are taken into critical consideration.

Keywords: Nucleobase; Peptide; Nucleopeptide; Nucleic acid

Structural analysis of the complex between calmodulin and full-length myelin basic protein, an intrinsically disordered molecule by Viivi Majava; Chaozhan Wang; Matti Myllykoski; Salla M. Kangas; Sung Ung Kang; Nobuhiro Hayashi; Peter Baumgärtel; Anthony M. Heape; Gert Lubec; Petri Kursula (pp. 59-71).

Myelin basic protein (MBP) is present between the cytoplasmic leaflets of the compact myelin membrane in both the peripheral and central nervous systems, and characterized to be intrinsically disordered in solution. One of the best-characterized protein ligands for MBP is calmodulin (CaM), a highly acidic calcium sensor. We pulled down MBP from human brain white matter as the major calcium-dependent CaM-binding protein. We then used full-length brain MBP, and a peptide from rodent MBP, to structurally characterize the MBP–CaM complex in solution by small-angle X-ray scattering, NMR spectroscopy, synchrotron radiation circular dichroism spectroscopy, and size exclusion chromatography. We determined 3D structures for the full-length protein–protein complex at different stoichiometries and detect ligand-induced folding of MBP. We also obtained thermodynamic data for the two CaM-binding sites of MBP, indicating that CaM does not collapse upon binding to MBP, and show that CaM and MBP colocalize in myelin sheaths. In addition, we analyzed the post-translational modifications of rat brain MBP, identifying a novel MBP modification, glucosylation. Our results provide a detailed picture of the MBP–CaM interaction, including a 3D model of the complex between full-length proteins.

Keywords: Myelin; Calmodulin; Protein complex; 3-dimensional structure; Myelin basic protein; Proteomics

Erratum to: Structural analysis of the complex between calmodulin and full-length myelin basic protein, an intrinsically disordered molecule by Viivi Majava; Chaozhan Wang; Matti Myllykoski; Salla M. Kangas; Sung Ung Kang; Nobuhiro Hayashi; Peter Baumgärtel; Anthony M. Heape; Gert Lubec; Petri Kursula (pp. 73-74).

Involvement of individual hippocampal signaling protein levels in spatial memory formation is strain-dependent by Sudarshan S. Patil; Florentine Schlick; Harald Höger; Gert Lubec (pp. 75-87).

Although a series of signaling cascades involved in spatial memory have been identified, their link to spatial memory and strain-dependent expression has not been reported so far. Hippocampal levels of the abovementioned signaling proteins were determined in laboratory inbred strain C57BL/6J, the wild-derived inbred strain PWD/PhJ and the wild caught mouse Apodemus sylvaticus (AS) by immunoblotting. The resulting hippocampal protein levels were correlated with results from MWM. Hippocampal signaling protein (hSP) levels were tested also in yoked controls. Within-strain comparison between trained and yoked controls revealed significant differences between levels of Phospho-CaMKII (alpha), Phospho-CREB, Egr-1, c-Src, Phospho-ERK5, Phospho-MEK5 and NOS1 in all of the three strains tested. In addition, the three strains revealed different involvement of individual hSP levels clearly indicating that individual mouse strains were linked to individual hSPs in spatial memory. Phospho-ERK5 levels were not detectable in hippocampi of yoked controls of each strain. We learn from this study that a series of hSPs are associated with spatial memory and that different hSPs are linked to spatial memory in different strains that show different outcome in the MWM. Even correlational patterns in the individual hSPs differed between mouse strains. This is of importance for the interpretation of previous studies on the abovementioned signaling cascades as well as for the design of future studies on these hippocampal proteins. It is intriguing that individual mouse strains, laboratory or wild caught, may use different signaling pathways for spatial memory in the Morris water maze.

Keywords: Apodemus sylvaticus ; Morris water maze; PWD/PhJ; Western blotting; Signaling proteins

Taurine inhibits osteoclastogenesis through the taurine transporter by Ling-Qing Yuan; Wei Liu; Rong-Rong Cui; Dan Wang; Ji-Cai Meng; Hui Xie; Xian-Ping Wu; Hou-De Zhou; Ying Lu; Er-Yuan Liao (pp. 89-99).

Several studies have suggested a direct link between taurine and bone homeostasis. However, the mechanisms of taurine on the regulation of bone metabolism have not been elucidated. Using a coculture of osteoblasts and bone marrow cells as a model for the study of osteoclastogenesis, RANKL-stimulated RAW264.7 cells and M-CSF- and RANKL-induced bone marrow macrophages were investigated to elucidate the possible roles of taurine in osteoclastogenesis. Taurine inhibited osteoclastogenesis in the coculture of osteoblasts and bone marrow cells, but did not influence the expression of OPG and RANKL in osteoblasts. The taurine transporter (TAUT) expressed by RAW264.7 and bone marrow macrophages exhibited typical taurine uptake activity. Taurine directly reduced osteoclastogenesis in RANKL-stimulated RAW264.7 cells and M-CSF- and RANKL-induced bone marrow macrophages, while TAUT siRNA relieved this effect. Our study demonstrated that taurine directly inhibited osteoclastogenesis through the taurine transporter. Taken together, these data suggest that taurine plays a direct role in bone homeostasis by inhibiting osteoclastogenesis.

Keywords: Taurine; Taurine transporter; Osteoclastogenesis; siRNA

Prediction of mitochondrial proteins of malaria parasite using split amino acid composition and PSSM profile by Ruchi Verma; Grish C. Varshney; G. P. S. Raghava (pp. 101-110).

The rate of human death due to malaria is increasing day-by-day. Thus the malaria causing parasite Plasmodium falciparum (PF) remains the cause of concern. With the wealth of data now available, it is imperative to understand protein localization in order to gain deeper insight into their functional roles. In this manuscript, an attempt has been made to develop prediction method for the localization of mitochondrial proteins. In this study, we describe a method for predicting mitochondrial proteins of malaria parasite using machine-learning technique. All models were trained and tested on 175 proteins (40 mitochondrial and 135 non-mitochondrial proteins) and evaluated using five-fold cross validation. We developed a Support Vector Machine (SVM) model for predicting mitochondrial proteins of P. falciparum, using amino acids and dipeptides composition and achieved maximum MCC 0.38 and 0.51, respectively. In this study, split amino acid composition (SAAC) is used where composition of N-termini, C-termini, and rest of protein is computed separately. The performance of SVM model improved significantly from MCC 0.38 to 0.73 when SAAC instead of simple amino acid composition was used as input. In addition, SVM model has been developed using composition of PSSM profile with MCC 0.75 and accuracy 91.38%. We achieved maximum MCC 0.81 with accuracy 92% using a hybrid model, which combines PSSM profile and SAAC. When evaluated on an independent dataset our method performs better than existing methods. A web server PFMpred has been developed for predicting mitochondrial proteins of malaria parasites ( http://www.imtech.res.in/raghava/pfmpred/ ).

Keywords: Plasmodium falciparum ; Mitochondria; Support vector machine; Position specific scoring matrix; Online server

Potential therapeutic radiotracers: preparation, biodistribution and metabolic characteristics of ¹⁷⁷Lu-labeled cyclic RGDfK dimer by Jiyun Shi; Zhaofei Liu; Bing Jia; Zilin Yu; Huiyun Zhao; Fan Wang (pp. 111-120).

In this study, we reported the preparation and evaluation of ¹⁷⁷Lu-DOTA-RGD2, ¹⁷⁷Lu-DOTA-Bz-RGD2 and ¹⁷⁷Lu-DTPA-Bz-RGD2 (RGD2 = E[c(RGDfK)]₂) as a potential therapeutic radiotracers for the treatment of integrin α_vβ₃-positive tumors. The BALB/c nude mice bearing the U87MG human glioma xenografts were used to evaluate the biodistribution characteristics and excretion kinetics of ¹⁷⁷Lu-DOTA-RGD2, ¹⁷⁷Lu-DOTA-Bz-RGD2 and ¹⁷⁷Lu-DTPA-Bz-RGD2. It was found that there were no major differences in their lipophilicity and biodistribution characteristics, particularly at latter time points. A major advantage of using DTPA-Bz as the bifunctional chelator (BFC) was its high radiolabeling efficiency (fast and high yield radiolabeling) at room temperature. Using DOTA and DOTA-Bz as BFCs, the radiolabeling kinetics was slow, and heating at 100°C and higher DOTA-conjugate concentration were needed for successful ¹⁷⁷Lu-labeling. Therefore, DTPA-Bz is an optimal BFC for routine preparation of ¹⁷⁷Lu-labeled cyclic RGDfK peptides, and ¹⁷⁷Lu-DTPA-Bz-RGD2 is worthy of further investigation for targeted radiotherapy of integrin α_vβ₃-positive tumors.

Keywords: Integrin α_vβ₃ ; RGD; DTPA-Bz; ¹⁷⁷Lu; Tumor therapy

2-Oxo-2H-benzo[h]benzopyran as a new light sensitive protecting group for neurotransmitter amino acids by Ana M. S. Soares; Susana P. G. Costa; M. Sameiro T. Gonçalves (pp. 121-133).

Aiming at the development of new benzopyran-based photocleavable protecting groups, novel chloromethylated and hydroxymethylated 2-oxo-2H-benzo[h]benzopyran derivatives bearing a methoxy substituent were designed and used in the synthesis of a series of fluorescent bioconjugates, by linking through an ester or urethane bond to several model neurotransmitter amino acids (glycine, alanine, β-alanine and γ-aminobutyric acid, GABA). The resulting fluorescent bioconjugates with emission in the visible range and high fluorescent quantum yields, were subjected to photocleavage reaction in methanol/HEPES buffer (80:20) solution at different wavelengths of irradiation (250, 300, 350 and 419 nm) and photocleavage kinetic data were obtained.

Keywords: Oxobenzobenzopyran; Coumarin; Neurotransmitters; Amino acids; Photocleavable protecting groups

LyeTx I, a potent antimicrobial peptide from the venom of the spider Lycosa erythrognatha by D. M. Santos; R. M. Verly; D. Piló-Veloso; M. de Maria; M. A. R. de Carvalho; P. S. Cisalpino; B. M. Soares; C. G. Diniz; L. M. Farias; D. F. F. Moreira; F. Frézard; M. P. Bemquerer; A. M. C. Pimenta; M. E. de Lima (pp. 135-144).

LyeTx I, an antimicrobial peptide isolated from the venom of Lycosa erythrognatha, known as wolf spider, has been synthesised and its structural profile studied by using the CD and NMR techniques. LyeTx I has shown to be active against bacteria (Escherichia coli and Staphylococcus aureus) and fungi (Candida krusei and Cryptococcus neoformans) and able to alter the permeabilisation of l-α-phosphatidylcholine-liposomes (POPC) in a dose-dependent manner. In POPC containing cholesterol or ergosterol, permeabilisation has either decreased about five times or remained unchanged, respectively. These results, along with the observed low haemolytic activity, indicated that antimicrobial membranes, rather than vertebrate membranes seem to be the preferential targets. However, the complexity of biological membranes compared to liposomes must be taken in account. Besides, other membrane components, such as proteins and even specific lipids, cannot be discarded to be important to the preferential action of the LyeTx I to the tested microorganisms. The secondary structure of LyeTx I shows a small random-coil region at the N-terminus followed by an α-helix that reached the amidated C-terminus, which might favour the peptide-membrane interaction. The high activity against bacteria together with the moderate activity against fungi and the low haemolytic activity have indicated LyeTx I as a good prototype for developing new antibiotic peptides.

Keywords: Lycosa erythrognatha ; Antimicrobial peptide; LyeTx I; Spider venom

The effects of a low protein diet on amino acids and enzymes in the serine synthesis pathway in mice by Jordan E. Antflick; Glen B. Baker; David Richard Hampson (pp. 145-153).

l-Serine is required for cellular and tissue growth and is particularly important in the immature brain where it acts as a crucial neurotrophic factor. In this study, the levels of amino acids and enzymes in the l-serine biosynthetic pathway were examined in the forebrain, cerebellum, liver, and kidney after the exposure of mice to protein-restricted diets. The levels of l-serine, d-serine, and l-serine-O-phosphate were quantified by HPLC and quantitative Western blotting was used to measure changes in protein levels of five enzymes in the pathway. The l-serine biosynthetic enzyme phosphoserine phosphatase was strongly upregulated, while the serine degradative enzymes serine racemase and serine dehydratase were downregulated in the livers and kidneys of mice fed low (6%) or very low (2%) protein diets for 2 weeks compared with mice fed a normal diet (18% protein). No changes in these enzymes were seen in the brain. The levels of l-serine increased in the livers of mice fed 2% protein; in contrast, d-serine levels were reduced below the limit of detection in the livers of mice given either the 6 or 2% diets. d-Serine is a co-agonist at the NMDA class of glutamate receptors; no alterations in NMDA-R1 subunit expression were observed in liver or brain after protein restriction. These findings demonstrate that the expression of l-serine synthetic and degradative enzymes display reciprocal changes in the liver and kidney to increase l-serine and decrease d-serine levels under conditions of protein restriction, and that the brain is insulated from such changes.

Keywords: l-Phosphoserine; l-Serine-O-phosphate; Protein restriction; N-methyl-d-aspartate; Phosphoserine phosphatase; Serine racemase; Taurine

CSD mRNA expression in rat testis and the effect of taurine on testosterone secretion by Jiancheng Yang; Gaofeng Wu; Ying Feng; Changmian Sun; Shumei Lin; Jianmin Hu (pp. 155-160).

In the present study, the cysteine sulfinate decarboxylase (CSD) mRNA expression was detected in rat testis by RT-PCR. The results showed that CSD mRNA was expressed in rat testis, and the putative encoded-amino acid sequence was exactly the same as that in rat liver which was already known. At the same time, the effects of taurine on testosterone secretion were investigated both in vivo and in vitro. In vivo, taurine were administered to male rats by tap water. The results showed that taurine obviously stimulated the secretion of FSH, LH and testosterone in serum, but showed no significant effect on the secretion of estradiol. Taurine administered in water could significantly increase the concentration of taurine in the blood and testis of rats. In vitro, cultured Leydig cells were treated with taurine independently or incubated with human chorionic gonadotropin (HCG) and progesterone. The results showed that taurine had biphasic effects on basal testosterone secretion in cultured Leydig cells. Low concentrations of taurine (0.1–100 μg/ml) could stimulate testosterone secretion, whereas high concentration of taurine (400 μg/ml) could inhibit testosterone secretion. Testosterone secretion stimulated by HCG was significantly increased by 10 and 100 μg/ml of taurine administration, and obviously decreased by treating with 400 μg/ml of taurine. Testosterone secretion induced by progesterone was significantly stimulated by treating with 1.0 and 10 μg/ml of taurine, however, it was significantly inhibited when treated with 400 μg/ml of taurine. Meanwhile, the effect of silencing CSD mRNA by siRNA on testosterone secretion was analyzed. The results showed that testosterone secretion was obviously decreased after the inhibition of CSD mRNA expression in cultured Leydig cells. These results indicated that taurine can be synthesized in rat testis by CSD pathway, and it plays important roles in testosterone secretion both in vivo and in vitro which need to be further investigated.

Keywords: Taurine; CSD mRNA expression; Testosterone secretion; Leydig cell; Rat

Synthesis of orthogonally protected l-threo-β-ethoxyasparagine by Jan Spengler; Marta Pelay; Judit Tulla-Puche; Fernando Albericio (pp. 161-165).

Orthogonally protected l-threo-β-ethoxyasparagine (Fmoc-EtOAsn(Trt)-OH, 1) was synthesized from diethyl (2S,3S)-2-azido-3-hydroxysuccinate 2 in eight steps as a building block for solid-phase peptide synthesis. The starting material is easily available in multi-gram scale from d-diethyltartrate. The transformation steps reported here are robust and scalable. Thus, a significant amount of 1 (1.8 g) was obtained in 21% overall yield. The synthesis reported is also expected to be useful for the preparation of other O-substituted l-threo-β-hydroxyasparagine derivatives.

Keywords: Hexafluoroacetone; Tritylamide; 2,4,6-Trimethoxybenzylamide; Amino acids; Peptide

Partitioning of acidic, basic and neutral amino acids into imidazolium-based ionic liquids by Ghodratollah Absalan; Morteza Akhond; Leila Sheikhian (pp. 167-174).

In this paper, partitioning behaviors of typical neutral (Alanine), acidic (Glutamic acid) and basic (Lysine) amino acids into imidazolium-based ionic liquids [C₄mim][PF₆], [C₆mim][PF₆], [C₈mim][PF₆], [C₆mim][BF₄] and [C₈mim][BF₄] as extracting solvents were examined. [C₆mim][BF₄] showed the best efficiency for partitioning of amino acids. The partition coefficients of amino acids in ionic liquids were found to depend strongly on pH of the aqueous solution, amino acid and ionic liquid chemical structures. Different chemical forms of amino acids in aqueous solutions were pH dependent, so the pH value of the aqueous phase was a determining factor for extraction of amino acids into ionic liquid phase. Both water content of ionic liquids and charge densities of their anionic and cationic parts were important factors for partitioning of cationic and anionic forms of amino acids into ionic liquid phase. Extracted amino acids were back extracted into phosphate buffer solutions adjusted on appropriate pH values. The results showed that ionic liquids could be used as suitable modifiers on the stationary phase of an HPLC column for efficient separation of acidic, basic, and neutral amino acids.

Keywords: Neutral amino acid; Acidic amino acid; Basic amino acid; Ionic liquid; Partition coefficient; Back extraction

Synthesis of a new family of 2-ethylidene-γ-unsaturated δ-amino esters via microwave activated Stille coupling by Gabriella Barozzino Consiglio; Francesca Gaggini; Alessandro Mordini; Gianna Reginato (pp. 175-180).

A simple approach to a new family of enantiomerically enriched polyunsaturated t-Boc-protected-δ-amino esters is described, via microwave promoted Stille coupling of (Z)-methyl-2-bromobutenoate with stannylated allylamines. The reaction conditions are mild and selective and disclose a simple way to 1-substituted butenoates of defined geometry.

Keywords: Amino acids; δ-Amino esters; Stille coupling; Acrylates; Microwave

Crucial roles of membrane stability and its related proteins in the tolerance of peach fruit to chilling injury by Changfeng Zhang; Zhansheng Ding; Xiangbing Xu; Qing Wang; Guozheng Qin; Shiping Tian (pp. 181-194).

Proteome patterns in peach fruit (Prunus persica L.) stored at different low temperatures were examined in order to gain a better understanding why peach fruit is less prone to chilling injury when stored at 0°C than at 5°C. Some differently expressed proteins in peach fruit stored at 0 and 5°C were identified using electrospray ionization quadrupole time-of-flight tandem mass spectrometry. Among these proteins, four membrane stability related proteins, i.e., enolase, temperature-induced lipocalin, major allergen Pru p 1, and type II SK2 dehydrin were enhanced, but three proteins related to phenolic compounds metabolization, cinnamyl-alcohol dehydrogenase 5, cinnamyl-alcohol dehydrogenase 1, and chorismate mutase, were repressed in peach fruit at 0°C as compared to that at 5°C. The abundance of glucose-6-phosphate dehydrogenase, NADP-dependent isocitrate dehydrogenase, and NADP-denpendent malic enzyme, which catalyze the reactions during sugar metabolism and energy pathways, was found to decrease in peach fruit stored at 0°C. In addition, our data revealed that low temperature of 0°C might regulate the endogenous H₂O₂ level, resulting in activating the transcriptional level of genes encoding the proteins related to membrane stability. These results provide a comprehensive knowledge to understand the mechanisms by which peach fruit stored at 0°C showed a higher chilling tolerance than that at 5°C.

Keywords: Chilling stress; Hydrogen peroxide; Membrane stability; Peach fruit; Proteomics

Functional interactions among STIM1, Orai1 and TRPC1 on the activation of SOCs in HL-7702 cells by Zhen-Ya Zhang; Li-Jie Pan; Zong-Ming Zhang (pp. 195-204).

STIM1, Orai1 and TRPC1 are all reported to be important for store-operated Ca²⁺ entry (SOCE) in diverse cells. However, there is no evidence for the functional interaction of the three proteins in SOCE in human liver cells. The objective of this study is to determine whether they are involved in SOCE in normal human liver cells. Liposomal transfection method was used to increase expression levels of the three proteins in HL-7702 cells, a normal human liver cell line. Western blot and single cell RT–PCR were applied to evaluate transfection effectiveness. Changes in store-operated current (I_SOC) and SOCE were investigated using whole-cell patch-clamp recording and calcium imaging. I_SOC is detected in HL-7702 cells and it is inhibited either by 2-Aminoethoxydiphenyl borate (2-APB) or La³⁺. Overexpression of STIM1 or Orai1 alone did not induce any change in I_SOC. TRPC1-transfection, however, caused approximate 2.5-fold increase in I_SOC. A large increase (>10-fold) in I_SOC emerged when both STIM1 and Orai1 were co-transfected into HL-7702 cells. Co-overexpression of STIM1 + TRPC1 also caused >10-fold increase in I_SOC, and addition of Orai1 did not cause any further increase. In HL-7702 cells, TRPC1 and Orai1 take part in SOCE independently of each other. Functional interactions of STIM1 and Orai1 or TRPC1 contribute to I_SOC activation.

Keywords: Store-operated channels; STIM1; Orai1; TRPC1; Human liver cells

The involvement of NMDA and AMPA receptors in the mechanism of antidepressant-like action of zinc in the forced swim test by B. Szewczyk; E. Poleszak; M. Sowa-Kućma; A. Wróbel; S. Słotwiński; J. Listos; P. Wlaź; A. Cichy; A. Siwek; M. Dybała; K. Gołembiowska; A. Pilc; Gabriel Nowak (pp. 205-217).

Antidepressant-like activity of zinc in the forced swim test (FST) was demonstrated previously. Enhancement of such activity by joint administration of zinc and antidepressants was also shown. However, mechanisms involved in this activity have not yet been established. The present study examined the involvement of the NMDA and AMPA receptors in zinc activity in the FST in mice and rats. Additionally, the influence of zinc on both glutamate and aspartate release in the rat brain was also determined. Zinc-induced antidepressant-like activity in the FST in both mice and rats was antagonized by N-methyl-d-aspartic acid (NMDA, 75 mg/kg, i.p.) administration. Moreover, low and ineffective doses of NMDA antagonists (CGP 37849, L-701,324, d-cycloserine, and MK-801) administered together with ineffective doses of zinc exhibit a significant reduction of immobility time in the FST. Additionally, we have demonstrated the reduction of immobility time by AMPA receptor potentiator, CX 614. The antidepressant-like activity of both CX 614 and zinc in the FST was abolished by NBQX (an antagonist of AMPA receptor, 10 mg/kg, i.p.), while the combined treatment of sub-effective doses of zinc and CX 614 significantly reduces the immobility time in the FST. The present study also demonstrated that zinc administration potentiated a veratridine-evoked glutamate and aspartate release in the rat’s prefrontal cortex and hippocampus. The present study further suggests the antidepressant properties of zinc and indicates the involvement of the NMDA and AMPA glutamatergic receptors in this activity.

Keywords: Forced swim test; Antidepressant; Zinc; NMDA; AMPA; Glutamate receptors

Intramuscular adaptations to eccentric exercise and antioxidant supplementation by Chad M. Kerksick; Richard B. Kreider; Darryn S. Willoughby (pp. 219-232).

Prophylactic supplementation of N-acetyl-cysteine (NAC) and epigallocatechin gallate (EGCG) was studied for physiological and cellular changes in skeletal muscle after eccentric muscle contractions. Thirty healthy, active males (20.0 ± 1.8 years, 160 ± 7.1 cm, 76.1 ± 17.0 kg) ingested for 14 days either 1,800 mg of NAC, 1,800 mg of EGCG, or 1,000 mg of fiber (glucomannan) placebo (PLC) in a double blind, prophylactic fashion. Subjects completed one eccentric exercise bout (100 repetitions at 30°/s) using the dominant knee extensors. Strength and soreness were assessed, and blood and muscle samples obtained before and 6, 24, 48, and 72 h with no muscle sample being collected at 72 h. Separate mixed factorial repeated measures ANOVA (P < 0.05) were used for all statistical analysis. All groups experienced significantly reduced peak torque production after 6 and 24 h, increased soreness at all time points from baseline [with even greater soreness levels 24 h after exercise in PLC when compared to EGCG and NAC (P < 0.05)], increased lactate dehydrogenase at 6 h, and increased creatine kinase 6, 24 and 48 h after exercise. No significant group × time interaction effects were found for serum cortisol, neutrophil counts, and the neutrophil:lymphocyte ratio; although, all values experienced significant changes 6 h after exercise (P < 0.05), but at no other time points. At 48 h after the exercise bout the Neu:Lym ratio in EGCG was significantly less than NAC (P < 0.05), whereas there was a trend (P = 0.08) for the EGCG values to be less when compared to PLC at this time point. Markers of intramuscular mitochondrial and cytosolic apoptosis were assessed (e.g., bax, bcl-2, cytochrome C, caspase-3 content/enzyme activity, and total DNA content). Significant increases (P < 0.05) in muscle levels of bax and bcl-2 were observed in all groups with no significant differences between groups, whereas no changes (P > 0.05) were reported for cytochrome C, caspase-3 content, caspase-3 enzyme activity, and total DNA. Caspase-3 enzyme activity was significantly greater in all groups 48 h after exercise when compared to baseline (P < 0.05) and 6 h (P < 0.05) after exercise. An eccentric bout of muscle contractions appears to significantly increase muscle damage, markers of mitochondrial apoptosis, apoptotic enzyme activity, and whole-blood cell markers of inflammation with no changes in oxidative stress. While soreness ratings were blunted in the two supplementation groups 24 h after exercise when compared to PLC values, more research is needed to determine the potential impact of EGCG and NAC supplementation on changes related to oxidative stress, apoptosis, and eccentric exercise.

Keywords: N-acetyl-cysteine; Catechin; Epigallocatechin gallate; Skeletal muscle; Damage

Mimetics of the disulfide bridge between the N- and C-terminal cysteines of the KLK3-stimulating peptide B-2 by Miikka Pakkala; Janne Weisell; Can Hekim; Jouko Vepsäläinen; Erik A. A. Wallen; Ulf-Håkan Stenman; Hannu Koistinen; Ale Närvänen (pp. 233-242).

Human prostate produces kallikrein-related peptidase 3 (KLK3, also known as prostate specific antigen), which is widely used as a prostate cancer marker. Proteolytically active KLK3 has been shown to inhibit angiogenesis and its expression decreases in poorly differentiated tumors. Thus, it may be possible to control prostate cancer growth with agents that stimulate the proteolytic activity of KLK3. We have earlier developed synthetic peptides, which bind specifically to KLK3 and promote its proteolytic activity. These peptides are cyclic, all containing a disulfide bridge between the N- and C-terminal cysteines. To increase the in vivo stability of the KLK3-stimulating peptide B-2, we made differently cyclized analogues by replacing both terminal cysteines and the disulfide bridge between them. A replacement consisting of γ-amino butyric acid and aspartic acid, where the amino group from the former was linked to the main chain carboxyl group of the latter, was found to be, at high concentrations, more active than the B-2 peptide. Furthermore, as compared to the parent peptide, this analog had an improved stability in plasma and against the enzymatic degradation by KLK3. In addition, the series of analogues also provided valuable information of the structure–activity relationships of the B-2 peptide.

Keywords: Synthetic peptide; Stability; Prostate cancer; Kallikrein-related peptidase 3; KLK3; Prostate specific antigen; PSA

Generation of in silico predicted coxsackievirus B3-derived MHC class I epitopes by proteasomes by Antje Voigt; Sandra Jäkel; Kathrin Textoris-Taube; Christin Keller; Ilse Drung; Gudrun Szalay; Karin Klingel; Peter Henklein; Karl Stangl; Peter M. Kloetzel; Ulrike Kuckelkorn (pp. 243-255).

Proteasomes are known to be the main suppliers of MHC class I (MHC-I) ligands. In an attempt to identify coxsackievirus B3 (CVB3)-MHC-I epitopes, a combined approach of in silico MHC-I/transporters associated with antigen processing (TAP)-binding and proteasomal cleavage prediction was applied. Accordingly, 13 potential epitopes originating from the structural and non-structural protein region of CVB3 were selected for further in vitro processing analysis by proteasomes. Mass spectrometry demonstrated the generation of seven of the 13 predicted MHC-I ligands or respective ligand precursors by proteasomes. Detailed processing analysis of three adjacent MHC-I ligands with partially overlapping sequences, i.e. VP2(273–281), VP2(284–292) and VP2(285–293), revealed the preferential generation predominantly of the VP2(285–293) epitope by immunoproteasomes due to altered cleavage site preferences. The VP2(285–293) peptide was identified to be a high affinity binder, rendering VP2(285–293) a likely candidate for CD8 T cell immunity in CVB3 infection. In conclusion, the concerted usage of different in silico prediction methods and in vitro epitope processing/presentation studies was supportive in the identification of CVB3 MHC-I epitopes.

Keywords: Proteasomes; Epitope prediction; Antigen processing; Coxsackievirus

Transglutaminase participates in the blockade of neurotransmitter release by tetanus toxin: evidence for a novel biological function by Francesco Facchiano; Florence Deloye; Frédéric Doussau; Giulio Innamorati; Anthony C. Ashton; J. Oliver Dolly; Simone Beninati; Angelo Facchiano; Alberto Luini; Bernard Poulain; Fabio Benfenati (pp. 257-269).

Inhibition of neuroexocytosis by tetanus neurotoxin (TeNT) involves VAMP-2/synaptobrevin-2 cleavage. However, deletion of the TeNT activity does not completely abolish its inhibitory action. TeNT is a potent activator of the cross-linking enzyme transglutaminase 2 (TGase 2) in vitro. The role of the latter mechanism in TeNT poisoning was investigated in isolated nerve terminals and intact neurons. TeNT-induced inhibition of glutamate release from rat cortical synaptosomes was associated with a simultaneous activation of neuronal transglutaminase (TGase) activity. The TeNT-induced blockade of neuroexocytosis was strongly attenuated by pretreatment of either live Aplysia neurons or isolated nerve terminals with specific TGase inhibitors or neutralizing antibodies. The same treatments completely abolished the residual blockade of neuroexocytosis of a non-proteolytic mutant of TeNT light chain. Electrophysiological studies indicated that TGase activation occurs at an early step of TeNT poisoning and contributes to the inhibition of transmitter release. Bioinformatics and biochemical analyses identified synapsin I and SNAP-25 as potential presynaptic TGase substrates in isolated nerve terminals, which are potentially involved in the inhibitory action of TeNT. The results suggest that neuronal TGase activity plays an important role in the regulation of neuroexocytosis and is one of the intracellular targets of TeNT in neurons.

Keywords: Neuroexocytosis; Transglutaminase 2; Tetanus toxin; VAMP/synaptobrevin-2; Synapsin I; SNAP-25; Melanoma; Proteomics

Wild type but not mutant APP is involved in protective adaptive responses against oxidants by Giovanna Cenini; Giuseppina Maccarinelli; Cristina Lanni; Sara Anna Bonini; Giulia Ferrari-Toninelli; Stefano Govoni; Marco Racchi; David Allan Butterfield; Maurizio Memo; Daniela Uberti (pp. 271-283).

This study points out different behaviour between HEK cells overexpressing wild-type or mutant APP when exposed to oxidative insult. Although apparently both APPwt and APPmut overexpression conferred resistance to oxidative insult, some differences in terms of degree of protection was observed in the two clones. We found that the two clones differed, especially, in terms of redox profile. HEK-APPmut cells were characterized by higher levels of oxidative markers in comparison with HEK-APPwt. In addition, SOD activity appeared more efficient in HEK-APPwt than in HEK-APPmut, thus justifying the differences in terms of cell survival in the two clones. We suggest that, according to “hormesis theory”, in HEK-APPwt cells low amount of oxidative stress can exert a beneficial effect that at a higher intensity results harmful. In contrast, HEK-APPmut cells lost this stress resistance probably because the degree of oxidative stress is too high and the antioxidant enzymes are themselves compromised.

Keywords: APP; p53; Oxidative stress; SOD; Cell vulnerability; Adaptive response

Profiling of residue-level photo-oxidative damage in peptides by Anita J. Grosvenor; James D. Morton; Jolon M. Dyer (pp. 285-296).

Protein and peptide oxidation is a key feature in the progression of a variety of disease states and in the poor performance of protein-based products. The present work demonstrates a mass spectrometry-based approach to profiling degradation at the amino acid residue level. Synthetic peptides containing the photosensitive residues, tryptophan and tyrosine, were used as models for protein-bound residue photodegradation. Electrospray ionisation tandem mass spectrometry (ESI-MS/MS) was utilised to characterise and provide relative quantitative information on the formation of photoproducts localised to specific residues, including the characterisation of low abundance photomodifications not previously reported, including W + 4O modification, hydroxy-bis-tryptophandione and topaquinone. Other photoproducts observed were consistent with the formation of tyrosine-derived dihydroxyphenylalanine (dopa), trihydroxyphenylalanine, dopa-quinone and nitrotyrosine, and tryptophan-derived hydroxytryptophan, dihydroxytryptophan/N-formylkynurenine, kynurenine, hydroxyformylkynurenine, tryptophandiones, tetrahydro-β-carboline and nitrotryptophan. This approach combined product identification and abundance tracking to generate a photodegradation profile of the model system. The profile of products formed yields information on formative mechanisms. Profiling of product formation offers new routes to identify damage markers for use in tracking and controlling oxidative damage to polypeptides.

Keywords: Oxidation; Photodegradation; Photo-oxidation; Tryptophan; Tyrosine; Mass spectrometry

Thermodynamics of binding of regulatory ligands to tissue transglutaminase by Carlo M. Bergamini; Alessia Dondi; Vincenzo Lanzara; Monica Squerzanti; Carlo Cervellati; Katy Montin; Carlo Mischiati; Gianluca Tasco; Russel Collighan; Martin Griffin; Rita Casadio (pp. 297-304).

The transamidating activity of tissue transglutaminase is regulated by the ligands calcium and GTP, via conformational changes which facilitate or interfere with interaction with the peptidyl-glutamine substrate. We have analysed binding of these ligands by calorimetric and computational approaches. In the case of GTP we have detected a single high affinity site (K _D ≈ 1 μM), with moderate thermal effects suggestive that binding GTP involves replacement of GDP, normally bound to the protein. On line with this possibility no significant binding was observed during titration with GDP and computational studies support this view. Titration with calcium at a high cation molar excess yielded a complex binding isotherm with a number of “apparent binding sites” in large excess over those detectable by equilibrium dialysis (6 sites). This binding pattern is ascribed to occurrence of additional thermal contributions, beyond those of binding, due to the occurrence of conformational changes and to catalysis itself (with protein self-crosslinking). In contrast only one site for binding calcium with high affinity (K _D ≈ 0.15 μM) is observed with samples of enzyme inactivated by alkylation at the active site (to prevent enzyme crosslinkage and thermal effects of catalysis). These results indicate an intrinsic ability of tissue transglutaminase to bind calcium with high affinity and the necessity of careful reassessment of the enzyme regulatory pattern in relation to the concentrations of ligands in living cells, taking also in account effects of ligands on protein subcellular compartimentation.

Keywords: Transglutaminase; Binding of ligands; Calcium; GTP; Isothermal titration calorimetry

Stereoselective introduction of two chiral centers by a single diketoreductase: an efficient biocatalytic route for the synthesis of statin side chains by Xuri Wu; Lili Wang; Shuzhen Wang; Yijun Chen (pp. 305-308).

Statins, including atorvastatin (Lipitor^®), are the top-selling drugs in the world. The biocatalytic production of chiral side chains of statin drugs is of great interest to academia and industry. Stereoselective double reduction of a β,δ-diketo ester catalyzed by a diketoreductase offers a simple and efficient route for the preparation of statin side chains. Comparison of different cofactor regeneration systems resulted in an easy and cost-effective process for this enzymatic reduction.

Keywords: Biocatalysis; Chiral side chain; Diketoreductase; Oxidoreductase; Statin

2-(Phenylethyl)ammonium hydrogensquarate hemihydrate: crystal structure, solid-state IR-spectroscopic and theoretical characterization by Bojidarka B. Ivanova; Rüdiger W. Seidel; Tsonko Kolev; William Sheldrick; Michael Spiteller (pp. 309-314).

The title compound, 2-(phenylethyl)ammonium hydrogensquarate hemihydrate, was synthesized and structurally and spectroscopically characterized by a single crystal X-ray diffraction and solid-state polarized IR spectroscopy of oriented colloids in a nematic host. The crystal structure consists of two crystallographically independent 2-(phenylethyl)ammonium cations, joined in a 2D hydrogen-bonded network with hydrogensquarate anions and solvent water molecules. Surprisingly, the crystallographically non-equivalent cations exhibit differing pseudo T and G trans configurations.

Keywords: 2-(Phenylethyl)ammonium hydrogensquarate hemihydrate; Crystal structure; Solid-state linear polarized IR spectroscopy; Amino acid derivatives; Biogenic amines

Amino acid content and nectar choice by forager honeybees (Apis mellifera L.) by Michele Bertazzini; Piotr Medrzycki; Laura Bortolotti; Lara Maistrello; Giuseppe Forlani (pp. 315-318).

Dual choice feeding tests were performed to determine a preference of forager honeybees for specific amino acids. Artificial nectar containing proline was preferred over those containing only sugars. Nectar containing alanine was preferred on the first day, but preference was no longer significant thereafter. On the contrary, a negative response was found for serine. When the bees were given the choice between two nectars enriched with different compounds, proline was preferred above both alanine and serine, and alanine above serine.

Keywords: Amino acid content; Feeding preference; Honeybee; Nectar

Addendum to 11th International Congress on Amino Acids, Peptides and Proteins (pp. 319-320).

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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.39, #1)