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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.32, #4)

Absorption, tissue distribution, metabolism and elimination of taurine given orally to rats by D. W. Sved; J. L. Godsey; S. L. Ledyard; A. P. Mahoney; P. L. Stetson; S. Ho; N. R. Myers; P. Resnis; A. G. Renwick (pp. 459-466).

Three biodisposition studies with taurine were performed in male and female adult rats at dosages of 30 and 300 mg/kg. A single oral dose of ¹⁴C-taurine was rapidly absorbed, distributed to tissues and excreted unchanged in urine. Elimination of radioactivity from intracellular pools was slow. Pre-treatment of animals for 14 days with unlabelled taurine did not significantly affect the fate of ¹⁴C-taurine. At the higher dose there was more extensive excretion combined with a lower percentage of the dose in the carcass, indicating the possibility of saturation of the tubular reabsorption mechanism for taurine. Daily administration of unlabelled taurine for 14 days did not result in an increase in total taurine in the brain. The data indicate that exogenous taurine rapidly equilibrates with endogenous body pools and that any excess is rapidly eliminated by the kidneys.

Keywords: Keywords: Taurine – Toxicokinetics – Absorption – Tissue distribution – Elimination

Effects of resistance training and protein plus amino acid supplementation on muscle anabolism, mass, and strength by D. S. Willoughby; J. R. Stout; C. D. Wilborn (pp. 467-477).

This study examined 10 wks of resistance training and the ingestion of supplemental protein and amino acids on muscle performance and markers of muscle anabolism. Nineteen untrained males were randomly assigned to supplement groups containing either 20 g protein (14 g whey and casein protein, 6 g free amino acids) or 20 g dextrose placebo ingested 1 h before and after exercise for a total of 40 g/d. Participants exercised 4 times/wk using 3 sets of 6–8 repetitions at 85–90% of the one repetition maximum. Data were analyzed with two-way ANOVA (p < 0.05). The protein supplement resulted in greater increases in total body mass, fat-free mass, thigh mass, muscle strength, serum IGF-1, IGF-1 mRNA, MHC I and IIa expression, and myofibrillar protein. Ten-wks of resistance training with 20 g protein and amino acids ingested 1 h before and after exercise is more effective than carbohydrate placebo in up-regulating markers of muscle protein synthesis and anabolism along with subsequent improvements in muscle performance.

Keywords: Keywords: Resistance training – Insulin – Myosin heavy chain – IGF-1 – Amino acids

Crystallization and preliminary X-ray diffraction analysis of E. coli arginyl-tRNA synthetase in complex form with a tRNA^Arg by M. Zhou; A. Azzi; X. Xia; E.-D. Wang; S.-X. Lin (pp. 479-482).

Amino acids are building blocks of proteins, while aminoacyl-tRNA synthetases (aaRSs) catalyze the first reaction in such building: the biosynthesis of proteins. The E. coli arginyl-tRNA synthetase (ArgRS) has been crystallized in complex form with tRNA^Arg (B. stearothermophilus), at pH 5.6 using ammonium sulfate as a precipitating agent. Two crystal forms have been identified based on unit cell dimension. The complete data sets from both crystal forms have been collected with a primitive hexagonal space group. A data set of Form II crystals at 3.2 Å and 94% completeness has been obtained, with unit cell parameters a = b = 98.0 Å, c = 463.2 Å, and α = β = 90°, γ = 120°, being different from a = b = 110.8 Å, c = 377.8 Å for form I. The structure determination will demonstrate the interaction of these two macromolecules to understand the special mechanism of ArgRS that requires the presence of tRNA for amino acid activation. Such complex structure also provides a wide opening for inhibitor search using bioinformatics.

Keywords: Keywords: Amino acids – Arginyl-tRNA synthetase – tRNA^Arg – Protein-RNA complex – Macromolecular crystallization – Amino acid activation – Aminoacylation – Protein biosynthesis

Using ensemble classifier to identify membrane protein types by H.-B. Shen; K.-C. Chou (pp. 483-488).

Predicting membrane protein type is both an important and challenging topic in current molecular and cellular biology. This is because knowledge of membrane protein type often provides useful clues for determining, or sheds light upon, the function of an uncharacterized membrane protein. With the explosion of newly-found protein sequences in the post-genomic era, it is in a great demand to develop a computational method for fast and reliably identifying the types of membrane proteins according to their primary sequences. In this paper, a novel classifier, the so-called “ensemble classifier”, was introduced. It is formed by fusing a set of nearest neighbor (NN) classifiers, each of which is defined in a different pseudo amino acid composition space. The type for a query protein is determined by the outcome of voting among these constituent individual classifiers. It was demonstrated through the self-consistency test, jackknife test, and independent dataset test that the ensemble classifier outperformed other existing classifiers widely used in biological literatures. It is anticipated that the idea of ensemble classifier can also be used to improve the prediction quality in classifying other attributes of proteins according to their sequences.

Keywords: Keywords: Type-I – Type-II – Multi-pass transmembrane – Lipid-chain-anchored – GPI-anchored – Pseudo-amino acid composition – Ensemble classifier – Fusion – Voting

The effect of extractants on degradation of L-glutamate and L-arginine in the course of shaking and filtration at low temperature by P. Formánek; B. Klejdus; V. Vranová (pp. 489-491).

The effects of demineralized water (DEMI H₂O) and 0.5 M ammonium acetate (0.5 M AAc) on losses of L-glutamic acid and L-arginine in the course of shaking and filtration at low temperature (6 °C) were tested. The concentration of L-glutamic acid decreased by 6.3% in DEMI H₂O and by 4.9% in 0.5 M AAc, whereas the L-arginine concentration decreased by 6.0% (DEMI H₂O) and 10.7% (0.5 M AAc). We found a significantly (P < 0.05) higher degradation of L-arginine in 0.5 M AAc compared with that of DEMI H₂O.

Keywords: Keywords: Demineralized water – Ammonium acetate – Amino acids – Losses – Extraction

Predicting secretory protein signal sequence cleavage sites by fusing the marks of global alignments by D.-Q. Liu; H. Liu; H.-B. Shen; J. Yang; K.-C. Chou (pp. 493-496).

A newly synthesized secretory protein in cells bears a special sequence, called signal peptide or sequence, which plays the role of “address tag” in guiding the protein to wherever it is needed. Such a unique function of signal sequences has stimulated novel strategies for drug design or reprogramming cells for gene therapy. To realize these new ideas and plans, however, it is important to develop an automated method for fast and accurately identifying the signal sequences or their cleavage sites. In this paper, a new method is developed for predicting the signal sequence of a query secretory protein by fusing the results from a series of global alignments through a voting system. The very high success rates thus obtained suggest that the novel approach is very promising, and that the new method may become a useful vehicle in identifying signal sequence, or at least serve as a complementary tool to the existing algorithms of this field.

Keywords: Keywords: Signal peptide – Cleavage site – Global alignment – Needleman–Wunsch algorithm – Secretory protein

Amino acid and protein modification by oxygen and nitrogen species by F. Galli (pp. 497-499).

Protein tyrosine nitration in hydrophilic and hydrophobic environments by S. Bartesaghi; G. Ferrer-Sueta; G. Peluffo; V. Valez; H. Zhang; B. Kalyanaraman; R. Radi (pp. 501-515).

In this review we address current concepts on the biological occurrence, levels and consequences of protein tyrosine nitration in biological systems. We focused on mechanistic aspects, emphasizing on the free radical mechanisms of protein 3-nitrotyrosine formation and critically analyzed the restrictions for obtaining large tyrosine nitration yields in vivo, mainly due to the presence of strong reducing systems (e.g. glutathione) that can potently inhibit at different levels the nitration process. Evidence is provided to show that the existence of metal-catalyzed processes, the assistance of nitric oxide-dependent nitration steps and the facilitation by hydrophobic environments, provide individually and/or in combination, feasible scenarios for nitration in complex biological milieux. Recent studies using hydrophobic tyrosine analogs and tyrosine-containing peptides have revealed that factors controlling nitration in hydrophobic environments such as biomembranes and lipoproteins can differ to those in aqueous compartments. In particular, exclusion of key soluble reductants from the lipid phase will more easily allow nitration and lipid-derived radicals are suggested as important mediators of the one-electron oxidation of tyrosine to tyrosyl radical in proteins associated to hydrophobic environments. Development and testing of hydrophilic and hydrophobic probes that can compete with endogenous constituents for the nitrating intermediates provide tools to unravel nitration mechanisms in vitro and in vivo; additionally, they could also serve to play cellular and tissue protective functions against the toxic effects of protein tyrosine nitration.

Keywords: Keywords: Tyrosine nitration – Peroxynitrite – Nitrogen dioxide – Hemeperoxidases – Free radicals – Hydrophobic environments

Lipid nitration and formation of lipid-protein adducts: biological insights by A. Trostchansky; H. Rubbo (pp. 517-522).

Lipid-protein adducts are formed during oxidative and nitrative stress conditions associated with increasing lipid and protein oxidation and nitration. The focus of this review is the analysis of interactions between oxidative-modified lipids and proteins and how lipid nitration can modulate lipid-protein adducts formation. For this, two biologically-relevant models will be analysed: a) human low density lipoprotein, whose oxidation is involved in the early steps of atherogenesis, and b) α-synuclein/lipid membranes system, where lipid-protein adducts are being associated with the develop of Parkinson disease and other synucleinopathies.

Keywords: Keywords: Lipid oxidation – Nitrated lipid – Peroxynitrite – Lipid-protein adduct – Nitric oxide – Free radicals – α-Synuclein – Low density lipoprotein

Regulation of apoptosis by protein S-nitrosylation by J. B. Mannick (pp. 523-526).

S-nitrosylation/denitrosylation of critical cysteine residues on proteins serves as a redox switch that regulates the function of a wide array of proteins. A key signaling pathway that is regulated by S-nitrosylation is apoptotic cell death. Here we will review the proteins in apoptotic pathways that are known to be S-nitrosylated by endogenous NO production. The targets and functional consequences of S-nitrosylation during apoptosis are multifaceted, allowing cells to fine tune their response to apoptotic signals.

Keywords: Keywords: S-nitrosylation – Apoptosis – Nitric oxide – Caspases – GAPDH – NMDA – Thioredoxin, Ask1, FLIP

The nuclear proteasome and the degradation of oxidatively damaged proteins by P. Voss; T. Grune (pp. 527-534).

The accumulation of oxidized proteins is known to be linked to some severe neurodegenerative diseases like Alzheimer’s, Parkinson’s and Huntington’s disease. Furthermore, the aging process is also accompanied by an ongoing aggregation of misfolded and damaged proteins. Therefore, mammalian cells have developed potent degradation systems, which selectively degrade damaged and misfolded proteins. The proteasomal system is largely responsible for the removal of oxidatively damaged proteins form the cellular environment. Not only cytosolic proteins are prone to oxidative stress, also nuclear proteins are readily oxidized. The nuclear proteasomal system is responsible for the degradation of these proteins. This review is focused on the specific degradation of oxidized nuclear proteins, the role of the proteasome in this process and the regulation of the nuclear proteasomal system under oxidative conditions.

Keywords: Keywords: Protein oxidation – Protein degradation – Proteasome – Nucleus – Histone – PARP

EPR spin-trapping of protein radicals to investigate biological oxidative mechanisms by O. Augusto; S. Muntz Vaz (pp. 535-542).

Presently, free radicals and oxidants are considered to mediate from signaling circuits involved in physiology and pathology to cell and tissue injury. The elucidation of these many inter-related processes requires a better understanding of cellular oxidative mechanisms many of which are mediated by protein radicals. Here, we will discuss the potentialities of EPR spin-trapping of protein radicals to unravel oxidative mechanisms. An overview of the methodology and its application to identify protein residues that are the target of specific oxidants, characterize emerging oxidants, and discriminate radical from non radical mechanisms will be presented. The examples are based on work developed in our laboratories but will be discussed in a broad scenario to emphasize that simple experiments can provide relevant insights into the biological reactivity of known and emerging biological oxidants and into signaling mechanisms.

Keywords: Keywords: Oxidative mechanisms – Protein radicals – EPR – EPR spin-trapping

Sulfenic acid in human serum albumin by S. Carballal; B. Alvarez; L. Turell; H. Botti; B. A. Freeman; R. Radi (pp. 543-551).

Sulfenic acid (RSOH) is a central intermediate in both the reversible and irreversible redox modulation by reactive species of an increasing number of proteins involved in signal transduction and enzymatic pathways. In this paper we focus on human serum albumin (HSA), the most abundant plasma protein, proposed to serve antioxidant functions in the vascular compartment. Sulfenic acid in HSA has been previously detected using different methods after oxidation of its single free thiol Cys34 through one- or two-electron mechanisms. Since recent evidence suggests that sulfenic acid in HSA is stabilized within the protein environment, this derivative represents an appropriate model to examine protein sulfenic acid biochemistry, structure and reactivity. Sulfenic acid in HSA could be involved in mixed disufide formation, supporting a role of HSA-Cys34 as an important redox regulator in extracellular compartments.

Keywords: Keywords: Thiol – Human serum albumin – Sulfenic acid – Peroxynitrite – Hydrogen peroxide – Free radicals

Hallmarks of protein oxidative damage in neurodegenerative diseases: focus on Alzheimer’s disease by M. C. Polidori; H. R. Griffiths; E. Mariani; P. Mecocci (pp. 553-559).

The pathogenesis of several neurodegenerative diseases, including Alzheimer’s disease, has been linked to a condition of oxidative and nitrosative stress, arising from the imbalance between increased reactive oxygen species (ROS) and reactive nitrogen species (RNS) production and antioxidant defences or efficiency of repair or removal systems. The effects of free radicals are expressed by the accumulation of oxidative damage to biomolecules: nucleic acids, lipids and proteins. In this review we focused our attention on the large body of evidence of oxidative damage to protein in Alzheimer’s disease brain and peripheral cells as well as in their role in signalling pathways. The progress in the understanding of the molecular alterations underlying Alzheimer’s disease will be useful in developing successful preventive and therapeutic strategies, since available drugs can only temporarily stabilize the disease, but are not able to block the neurodegenerative process.

Keywords: Keywords: Alzheimer’s disease – Aging – Oxidative stress – Nitrosative stress – Protein

Mechanisms of homocysteine toxicity in humans by J. Perła-Kaján; T. Twardowski; H. Jakubowski (pp. 561-572).

Homocysteine, a non-protein amino acid, is an important risk factor for ischemic heart disease and stroke in humans. This review provides an overview of homocysteine influence on endothelium function as well as on protein metabolism with a special respect to posttranslational modification of protein with homocysteine thiolactone. Homocysteine is a pro-thrombotic factor, vasodilation impairing agent, pro-inflammatory factor and endoplasmatic reticulum-stress inducer. Incorporation of Hcy into protein via disulfide or amide linkages (S-homocysteinylation or N-homocysteinylation) affects protein structure and function. Protein N-homocysteinylation causes cellular toxicity and elicits autoimmune response, which may contribute to atherogenesis.

Keywords: Keywords: Homocysteine – Homocysteine thiolactone – Protein N-homocysteinylation – Toxicity – Autoantibodies – Protein S-homocysteinylation

Oxidatively-modified and glycated proteins as candidate pro-inflammatory toxins in uremia and dialysis patients by M. Piroddi; I. Depunzio; V. Calabrese; C. Mancuso; C. M. Aisa; L. Binaglia; A. Minelli; A. D. Butterfield; F. Galli (pp. 573-592).

End stage renal disease (ESRD) patients accumulate blood hallmarks of protein glycation and oxidation. It is now well established that these protein damage products may represent a heterogeneous class of uremic toxins with pro-inflammatory and pro-oxidant properties. These toxins could be directly involved in the pathogenesis of the inflammatory syndrome and vascular complications, which are mainly sustained by the uremic state and bioincompatibility of dialysis therapy. A key underlying event in the toxicity of these proteinaceous solutes has been identified in scavenger receptor-dependent recognition and elimination by inflammatory and endothelial cells, which once activated generate further and even more pronounced protein injuries by a self-feeding mechanism based on inflammation and oxidative stress-derived events. This review examines the literature and provides original information on the techniques for investigating proteinaceous pro-inflammatory toxins. We have also evaluated therapeutic – either pharmacological or dialytic – strategies proposed to alleviate the accumulation of these toxins and to constrain the inflammatory and oxidative burden of ESRD.

Keywords: Keywords: Protein damage – Oxidation – Glycation – AGEs – Inflammation – Reactive oxygen species – Nitric oxide – Uremia – Dialysis – Proteomics

Kinetic and proteomic analyses of S-nitrosoglutathione-treated hexokinase A: consequences for cancer energy metabolism by S. Miller; C. Ross-Inta; C. Giulivi (pp. 593-602).

Mammalian hexokinase (HXK) is found at the outer mitochondrial membrane, exposed to mitochondrial oxygen- and nitrogen-radicals. Given the important role of this enzyme in metabolic pathways and diseases, the effect of S-nitrosoglutathione (GSNO) on HXK A structure and activity was studied. To focus on the catalytic domain, yeast HXK A was used because it has a significant homology to the mammalian domain that contains both the regulatory and catalytic sites. Biologically relevant [GSNO]/[HXK] caused a significant decrease in V_max with glucose (but not with fructose), along with oxidation of 5 Met and nitration of 4 Tyr. Preincubation of HXK with glucose abrogated the effect of GSNO whereas fructose was ineffective. These results are interpreted by considering the tight binding of glucose to the enzyme as opposed to that of fructose. The segment comprised from amino acids 304 to 306 contained the most modifications. Given that this sequence is highly conserved in HXK from various species, a decline in activity is expected when a high-affinity substrate is presented.Considering that changes in primary structure are envisioned at high [GSNO]/[HXK] ratios, like those present under normal conditions, it could be hypothesized that the high concentration of hexokinase present in fast growing tumors may serve not only to sustain high glycolysis rates, but also to minimize protein damage that might result in activity decline, compromising energy metabolism.

Keywords: Keywords: Nitrosoglutathione – Hexokinase – Structure – Activity – Oxygen radicals – Nitrogen radicals – Proteomics – Tumor – Glycolysis

Protein-carbonyl accumulation in the non-replicative senescence of the methionine sulfoxide reductase A (msrA) knockout yeast strain by D. Oien; J. Moskovitz (pp. 603-606).

The major enzyme of the methionine sulfoxide reductase (Msr) system is MsrA. Senescing msrA knockout mother yeast cells accumulated significant amounts of protein-carbonyl both at 5 generation-old (young) and 21 generation-old (old) cultures, while the control mother cells showed significant levels of protein-carbonyl mainly in the old culture. The Msr activities of both yeast strains declined with age and exposure of cells to H₂O₂ caused an accumulation of protein-carbonyl especially in the msrA knockout strain. It is suggested that a compromised MsrA activity may serve as a marker for non-replicative aging.

Keywords: Keywords: Oxidative-stress – Non-replicative senescence – Post-translation modification

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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.32, #4)