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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.30, #1)

Cysteine S-conjugate β-lyases by A. J. L. Cooper; J. T. Pinto (pp. 1-15).

Cysteine S-conjugate β-lyases are pyridoxal 5′-phosphate-containing enzymes that catalyze β-elimination reactions with cysteine S-conjugates that possess an electron-withdrawing group attached at the sulfur. The end products of the β-lyase reaction are pyruvate, ammonium and a sulfur-containing fragment. If the sulfur-containing fragment is reactive, the parent cysteine S-conjugate may be toxic, particularly to kidney mitochondria. Halogenated alkenes are examples of electrophiles that are bioactivated (toxified) by conversion to cysteine S-conjugates. These conjugates are converted by cysteine S-conjugate β-lyases to thioacylating fragments. Several cysteine S-conjugates found in allium foods (garlic and onion) are β-lyase substrates. This finding may account in part for the chemopreventive activity of allium products. This review (1) identifies enzymes that catalyze cysteine S-conjugate β-lyase reactions, (2) suggests that toxicant channeling may contribute to halogenated cysteine S-conjugate-induced toxicity to mitochondria, and (3) proposes mechanisms that may contribute to the antiproliferative effects of sulfur-containing fragments eliminated from allium-derived cysteine S-conjugates.

Keywords: Keywords: Cysteine S-conjugates – Cysteine S-conjugate β-lyases – S-(1,2-dichlorovinyl)-L-cysteine – Glutamine transaminase K – Mitochon drial aspartate aminotransferase – S-(1,1,2,2-tetrafluoroethyl)-L-cysteine – Allium-derived compounds

Circular dichroism and cross-linking studies of bacteriorhodopsin mutants by E. Karnaukhova; K. L. Schey; R. K. Crouch (pp. 17-23).

Circular dichroism (CD) spectroscopy was employed for native (wild type, WT) bacteriorhodopsin (bR) and several mutant derivatives: R134K, R134H, R82Q, S35C, L66C, and R134C/E194C. Comparative analysis of the CD spectra in visible range shows that only R134C/E194C exhibits biphasic CD, typical for native bR, the other mutants demonstrate CD spectra with significantly smaller or absent negative band. Since the biphasic CD is a feature of hexagonal lattice structure composed by bR trimers in the purple membrane, these mutants and WT were examined by cross-linking studies, which confirmed the same trend towards trimeric organization. Therefore, a single amino acid substitution may lead to drastically different CD spectra without disruption of bR trimeric organization. Thus, although disruption of bR trimeric crystalline lattice structure (e.g., solubilization with detergents) directly results in the disappearance of characteristic bilobe in visible CD, the lack of the bilobe in the CD alone does not predict the absence of trimers.

Keywords: Keywords: Bacteriorhodopsin – Mutants – Circular dichroism – Cross-linking – Matrix-assisted laser desorption/ionization mass spectrometry

Synthesis, tandem MS- and NMR-based characterization, and quantification of the carbon 13-labeled advanced glycation endproduct, 6-N-carboxymethyllysine by T. Delatour; F. Fenaille; V. Parisod; F. Arce Vera; T. Buetler (pp. 25-34).

6-N-carboxymethyllysine (CML), generated by the glycation and/or oxidation of lysine residues, has been measured in biological materials and food products using techniques such as ELISA, HPLC with fluorescence detection and mass spectrometry methods. Only limited information has been reported regarding the preparation of standards labeled with either deuterium, ¹³C or ¹⁵N atoms to be used as internal standards. In the present paper, a synthesis of carbon-13 labeled CML is described using l,2-¹³C₂-glyoxylic acid and 2-N-acetyllysine as starting materials. The resulting labeled 2-N-acetyl-CML was purified by HPLC-UV as a dibutyl ester. After a deprotection step, the yield was evaluated to be 53% when the reaction was conducted 17 h at 37°C. CML was extensively studied by ¹H- and ¹³C-NMR and the fragments observed in the collision induced dissociation (CID) spectrum were also assigned. Finally, the standards of CML and carbon-13 labeled CML were accurately quantified based on ¹H-NMR and tandem MS using lysine as an internal reference.

Keywords: Keywords: CML – Synthesis – Stable isotope – MS – NMR

Characterisation of the thiol–disulphide chemistry of desmopressin by LC, μ-LC, LC-ESI-MS and Maldi-Tof by T. Schmitz; C. W. Huck; A. Bernkop-Schnürch (pp. 35-42).

To date, the majority of therapeutic peptides and proteins have to be administered via parenteral routes, which are painful and inconvenient. In order to gain sufficient high blood concentrations after oral application, various barriers in the gastrointestinal tract have to be overcome. Apart from a poor membrane uptake and intense enzymatic degradation, this study will demonstrate that thiol–disulphide reactions are an underestimated essential part of the presystemic metabolism. Glutathione, integrative part of the antioxidant defence system in the gastrointestinal tract, may play an important role in the inactivation of orally given peptides and proteins. In order to verify this hypothesis, desmopressin which bears a single disulphide bond was used as model peptide drug. Desmopressin was incubated with GSH in various concentrations, and the extent of thiol/disulphide exchange reactions between the peptide drug and GSH was investigated in dependence on pH and ratio of reactants determined as a function of time via HPLC, LC-MS and Maldi-Tof-MS analyses.Results showed that desmopressin is degraded by 1% reduced glutathione at pH 4 and pH 5.5. In presence of 0.01%, 0.1% and 1% of reduced glutathione 6.1%, 19.4% and 52.1% of desmopressin, respectively, were degraded. The masses of the conjugates after deconvolution measured by liquid chromatography and electrospray ionisation mass spectrometric detection were m/z 1069.67, m/z 1376.50, m/z 1683.40 and m/z 2138. These molecular masses, confirmed by Maldi-Tof-MS analysis, correspond with the masses of conjugates expected in theory. Under defined conditions, these results reveal that thiol–disulphide exchange reactions have a considerable impact on the alteration of peptide drugs and proteins.

Keywords: Keywords: Glutathione – Desmopressin – Thiol-disulphide exchange – Drug delivery – Peptides

Enhancing effect of taurine on CYP7A1 mRNA expression in Hep G2 cells by N. V. Lam; W. Chen; K. Suruga; N. Nishimura; T. Goda; H. Yokogoshi (pp. 43-48).

Taurine has been reported to enhance cholesterol 7α-hydroxylase (CYP7A1) mRNA expression in animal models. However, no in vitro studies of this effect have been reported. The Hep G2 human hepatoma cell line has been recognized as a good model for studying the regulation of human CYP7A1. This work characterizes the effects of taurine on CYP7A1 mRNA levels of Hep G2 cells in a dose- and time-dependent manner. In the dose-dependent experiment, Hep G2 cells were treated with 0, 2, 10 or 20 mM taurine in the presence or absence of cholesterol 0.2 mM for 48 h. In the time-dependent experiment, Hep G2 cells were treated with 0 or 20 mM taurine for 4, 24 and 48 h with and without cholesterol 0.2 mM. Our data revealed that taurine showed time- and dose-response effects on CYP7A1 mRNA levels in Hep G2 cells. However, glycine – a structural analogue of taurine – did not have an effect on CYP7A1 gene expression. These results show that, in agreement to previous studies on animal models, taurine induces the mRNA levels of CYP7A1 in Hep G2 cells, which could enhance cholesterol conversion into bile acids. Also, Hep G2 cell line may be an appropriate model to study the effects of taurine on human cholesterol metabolism.

Keywords: Keywords: Taurine – CYP7A1 – Hep G2 – Cholesterol

Using cellular automata images and pseudo amino acid composition to predict protein subcellular location by X. Xiao; S. Shao; Y. Ding; Z. Huang; K.-C. Chou (pp. 49-54).

The avalanche of newly found protein sequences in the post-genomic era has motivated and challenged us to develop an automated method that can rapidly and accurately predict the localization of an uncharacterized protein in cells because the knowledge thus obtained can greatly speed up the process in finding its biological functions. However, it is very difficult to establish such a desired predictor by acquiring the key statistical information buried in a pile of extremely complicated and highly variable sequences. In this paper, based on the concept of the pseudo amino acid composition (Chou, K. C. PROTEINS: Structure, Function, and Genetics, 2001, 43: 246–255), the approach of cellular automata image is introduced to cope with this problem. Many important features, which are originally hidden in the long amino acid sequences, can be clearly displayed through their cellular automata images. One of the remarkable merits by doing so is that many image recognition tools can be straightforwardly applied to the target aimed here. High success rates were observed through the self-consistency, jackknife, and independent dataset tests, respectively.

Keywords: Keywords: Cellular automata images – Pseudo amino-acid composition – Protein subcellular location – Complexity – Covariant-discriminant algorithm

Reaction of pyridoxamine with malondialdehyde: Mechanism of inhibition of formation of advanced lipoxidation end-products by Z. Kang; H. Li; G. Li; D. Yin (pp. 55-61).

Advanced glycation end products (AGEs) and advanced lipoxidation end products (ALEs) are implicated in many age-related chronic diseases and in protein aging. Recent studies suggest that pyridoxamine (PM) is an efficient AGEs/ALEs inhibitor in various biological systems. Because malondialdehyde (MDA) is an important intermediate in the formation of ALEs during lipid peroxidation, the purpose of this study is to determine whether PM can trap MDA directly and thereby prevent ALEs formation. PM reacted readily with MDA under physiological conditions. Within 6 h, a 1-pyridoxamino-propenal adduct derived from reaction of equimolar PM + MDA was detected. A 1-amino-3-iminopropene complex and a dihydropyridine-pyridinium complex were also identified after 7 d incubation. PM also greatly inhibited the lipofuscin-like fluorescence formation induced by MDA reaction with bovine serum albumin (BSA). Our results showed clearly that PM inhibited the formation of ALEs by trapping MDA directly under physiological condition, and provide insight into the mechanism of action of PM in protecting proteins against carbonyl stress.

Keywords: Keywords: Advanced glycation end products (AGEs) – Advanced lipoxidation end products (ALEs) – Carbonyl stress – Pyridoxamine (PM) – Malondialdehyde (MDA)

Identification of inducible protein complexes in the phenol degrader Pseudomonas sp. strain phDV1 by blue native gel electrophoresis and mass spectrometry by E. Tsirogianni; M. Aivaliotis; D. G. Papasotiriou; M. Karas; G. Tsiotis (pp. 63-72).

Pseudomonas sp. strain phDV1, being a phenol degrading bacterium, has been found to utilize phenol as sole carbon source via the meta pathway. Blue native polyacrylamide gel electrophoresis (BN-PAGE) is widely used for the analysis of oligomeric state and molecular mass non-dissociated protein complexes. In this study, a number of proteomic techniques were used to investigate the oligomeric state enzymes involved in the aromatic degradation pathway. In particular, the Pseudomonas sp. strain phDV1 proteome was monitored under two different growth substrate conditions, using glucose or phenol as sole carbon source. The protein complexes map was compared by BN-PAGE after fractionation by sucrose density centrifugation of the cell extracts. Multiple differences were detected. Further, analysis and identification of the subunit composition of these complexes was carried out using MALDI-TOF MS, allowing the identification of 49 proteins. Additionally, functional information regarding protein–protein interactions was assembled, by coupling 2-D BN-PAGE with MALDI-TOF MS. Application of this functional proteomics method resulted in an higher number of the identified proteins.

Keywords: Keywords: MALDI – Phenol degradation – Pseudomonas sp. strain phDV1 – Protein complex – Blue native

Functional characterization of brain mitochondrial nitric oxide synthase during hypertension and aging by E. Calderón-Cortés; M Clemente-Guerrero; E. Sierra-Campos; C. Cortés-Rojo; F. J. Gaona-Zamudio; R. Villalobos-Molina; A. Saavedra-Molina (pp. 73-80).

Nitric oxide (NO^•) plays an important role in various physiological processes. The aim of the present study was to investigate if brain mitochondrial nitric oxide synthase (mtNOS) is active and functional during hypertension. L-citrulline production, an indicator of nitric oxide synthesis, was concentration-dependent on L-arginine in all strains and all ages tested, and was inhibited by 7-Nitroindazole (7-NI). Brain mitochondria of 1 month-old (prehypertensive) spontaneously hypertensive rats (SHR) exhibited a significantly (p < 0.05) low basal L-citrulline content as compared to age-matched Wistar (W) and Wistar-Kyoto (WKY) rats. L-citrulline synthesis in SHR rats showed a significant (p < 0.01) low response to L-arginine in 3 and 7 months-old rats. Respiratory rates in states 3 and 4 increased with low L-arginine concentration in all strains and all ages. The results suggest that in rat brain mitochondria, L-citrulline synthesis is constant once age-related hypertension is installed and NO^• does not regulate oxidative phosphorylation.

Keywords: Keywords: Mitochondrial L-citrulline – Rat brain – Spontaneously hypertensive rats – Aging – L-Arginine – Nitric oxide synthase

Plasma arginine correlations in trauma and sepsis by C. Chiarla; I. Giovannini; J. H. Siegel (pp. 81-86).

Arginine (ARG) is an amino acid (AA) with unique properties and with a key-role in the metabolic, immune and reparative response to trauma and sepsis. This study has been performed to characterize the correlations between plasma levels of ARG, of other AA and of multiple metabolic variables in trauma and sepsis.Two-hundred and sixty-three plasma amino-acidograms with a large series of additional biochemical and blood variables were obtained consecutively in 9 trauma patients who developed sepsis, undergoing total parenteral nutrition with dextrose, fat and a mixed AA solution containing 10.4% arginine.ARG was low soon after trauma, then it increased with increasing distance from trauma and with the development of sepsis. ARG was also directly related to the AA infusion rate (AAIR) and for any given AAIR, was lower after trauma than after the development of sepsis. ARG was also related directly to the plasma levels of most of the other AA, the best correlation being that with lysine (r² = 0.81, p < 0.001). These correlations were often shifted downwards (showing lower ARG for any given level of the other AA) in measurements performed after trauma, compared to those performed after development of sepsis; this effect was more pronounced for the correlations with branched chain AA. Correlations between ARG and non-AA variables were not particularly relevant. The best simultaneous correlates of ARG, among variables involved in plasma ARG availability, were citrulline level, AAIR and urinary 3-methylhistidine excretion (accounting for the effect of endogenous proteolysis) (multiple r² = 0.70, p < 0.001). Plasma ornithine (ORN), the AA more specifically linked to ARG metabolism, correlated with AAIR better than ARG and, for any given AAIR, was lower after trauma than after the development of sepsis. Correlations of ORN with other AA levels were poorer than those found for ARG, however ORN was directly related to white blood cell and platelet count, fibrinogen, transferrin, cholesterol and many AA clearances.These data show that changes in ARG in trauma and sepsis are correlated with changes in other AA and, within these correlations, reconfirm a tendency to lower ARG in trauma compared to sepsis. The strong correlation with lysine warrants a deeper assessment of the practical implications of interdependency between these two AA. The data also suggest that changes in plasma ORN in trauma and sepsis may reflect adequacy of AA substrate to support acute-phase and other synthetic processes.

Keywords: Keywords: Plasma arginine – Amino acids – Sepsis – Parenteral nutrition – Ornithine – Branched chain amino acids

Relationship of taurine and other amino acids in plasma and in neutrophils of septic trauma patients by J. M. Engel; J. Mühling; S. Weiss; B. Kärcher; T. Löhr; T. Menges; S. Little; G. Hempelmann (pp. 87-94).

Recently, an interdependency of plasma taurine and other amino acids as well as metabolic and clinical variables implicating therapeutic options was reported. This result may be an indication that plasma taurine levels are directly related to intracellular levels. Therefore, the aim of this study was to analyse the possible relationship between taurine levels in plasma and in neutrophils, the relationship to other amino acids, and variables quantifying metabolic impairment and severity of sepsis in multiple trauma patients developing sepsis. After multiple trauma taurine decreased significantly in plasma in thirty-two patients as well as within the neutrophil and does not recover in sepsis. Lower individual levels in the neutrophil did not follow lower individual levels in plasma and no correlation of taurine in plasma and in the neutrophils could be observed. In sepsis, only plasma showed an interdependency of taurine, aspartate, and glutamate. No association between taurine plasma or intracellular levels and SOFA score as indicator for severity of sepsis or metabolic variables was observed. After multiple trauma and in sepsis, taurine uptake in cells (which is regulated in different ways), and intracellular taurine (which serves e.g. as an osmolyte) can be influenced. Therefore a prediction of the neutrophil taurine pool seems not fully possible from taurine plasma levels. Intracellular taurine has some unique properties explaining the missing interdependency despite some similarities in osmoregulation and metabolic interactions to other amino acids. The association of taurine, aspartate, and glutamate in plasma cannot be simply transferred to the neutrophils intracellular level. The clinical meaning of the plasma correlation remains unclear. A dependency of plasma and neutrophil taurine to severity of sepsis and to metabolic variables seems not possible because of the multifactorial pathophysiology of sepsis.

Keywords: Keywords: Taurine – Amino acids – Neutrophil – Sepsis

An efficient route from trifluoroacetates to water soluble free amines using Diaion^® HP-20 by A. Korda; Z. Wróbel; K. Gwardiak (pp. 95-98).

A series of polybasic lysine and ornithine derivatives were synthesised as trifluoroacetate salts. Attempts to prepare their free amines according to standard methodology were not successful due to the excellent water solubility of these compounds. Free amines were however efficiently obtained if the column with Diaion^® HP-20 adsorbent was loaded with the trifluoroacetate and 1% NaHCO₃ aq was passed, followed by elution of free amine with methanol.

Keywords: Keywords: Amino acid – Peptide – Diaion^® HP-20

A method for simple identification of signature peptides derived from polyUb-K48 and K63 by MALDI-TOF MS and chemically assisted MS/MS fragmentation by A. P. Døskeland (pp. 99-103).

A simple method is described to identify signature peptides derived from polyubiquitin (polyUb) chains. The method is based on MALDI-TOF MS/MS analysis after chemically assisted fragmentation, and works on peptides isolated from polyacrylamide gels. PolyUb chains branched at K48 and K63 were chosen as models for Ub-protein conjugates. They were resolved by SDS-PAGE, and their tryptic peptides (in-gel-trypsinolysis) derivatized with 3-sulfopropinic acid NHSester to obtain chemically assisted fragmentation during the MS/MS analysis. PolyUb-K63 produced a single peptide identified as ⁵⁵TLSDYNIQK⁶³ (GG)ESTLHLVLR⁷². PolyUb-K48 produced two branched signature peptides identified as ⁴³LIFAGK⁴⁸(GG)QLEDGR⁵⁴ and ⁴³LIFAGK⁴⁸(LRGG)QLEDGR⁵⁴. The recovery of signature peptide with LRGG as branched chain underscores the need to take limited proteolysis into account in the search for detection of ubiquitinated peptides in proteomics studies. In conclusion, a simple method has been described allowing the identification of signature peptides, which are diagnostic markers of the majority of polyUb-conjugated proteins. In principle, the method should be applicable also for other more rare signature peptides.

Keywords: Keywords: MALDI-TOF MS/MS – Chemically assisted fragmentation – Polyubiquitin chains – Isopeptides – Signature peptides

14-3-3 proteins are involved in the regulation of mammalian cell proliferation by M. Rosner; M. Hengstschläger (pp. 105-109).

The 14-3-3 proteins are a family of abundant, widely expressed acidic polypeptides. The seven isoforms interact with over 70 different proteins. 14-3-3 isoforms have been demonstrated to be involved in the control of positive as well as negative regulators of mammalian cell proliferation. Here we used the approach of inactivating 14-3-3 protein functions via overexpression of dominant negative mutants to analyse the role of 14-3-3 proteins in mammalian cell proliferation. We found 14-3-3 dominant negative mutants to downregulate the proliferation rates of HeLa cells. Overexpression of these dominant negative mutants triggers upregulation of the protein levels of the cyclin-dependent kinase inhibitor p27, a major negative cell cycle regulator. In addition, they downregulate the protein levels of the important cell cycle promoter cyclin D1. These data provide new insights into mammalian cell proliferation control and allow a better understanding of the functions of 14-3-3 proteins.

Keywords: Keywords: 14-3-3 – Dominant negative mutants – Cell proliferation

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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.30, #1)