Check out our New Publishers' Select for Free Articles

Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.42, #1)

Amino acid and protein modification by oxygen and nitrogen species by Francesco Galli (pp. 1-4).

Protein oxidation: role in signalling and detection by mass spectrometry by Corinne M. Spickett; Andrew R. Pitt (pp. 5-21).

Proteins can undergo a wide variety of oxidative post-translational modifications (oxPTM); while reversible modifications are thought to be relevant in physiological processes, non-reversible oxPTM may contribute to pathological situations and disease. The oxidant is also important in determining the type of oxPTM, such as oxidation, chlorination or nitration. The best characterized oxPTMs involved in signalling modulation are partial oxidations of cysteine to disulfide, glutathionylated or sulfenic acid forms that can be reversed by thiol reductants. Proline hydroxylation in HIF signalling is also quite well characterized, and there is increasing evidence that specific oxidations of methionine and tyrosine may have some biological roles. For some proteins regulated by cysteine oxidation, the residues and molecular mechanism involved have been extensively studied and are well understood, such as the protein tyrosine phosphatase PTP1B and MAP3 kinase ASK1, as well as transcription factor complex Keap1–Nrf2. The advances in understanding of the role oxPTMs in signalling have been facilitated by advances in analytical technology, in particular tandem mass spectrometry techniques. Combinations of peptide sequencing by collisionally induced dissociation and precursor ion scanning or neutral loss to select for specific oxPTMs have proved very useful for identifying oxidatively modified proteins and mapping the sites of oxidation. The development of specific labelling and enrichment procedures for S-nitrosylation or disulfide formation has proved invaluable, and there is ongoing work to establish analogous methods for detection of nitrotyrosine and other modifications.

Keywords: Redox signalling; Nitrotyrosine; Oxidative stress; Precursor ion scanning; Cysteine oxidation

Oxidative protein damage and the proteasome by S. Grimm; A. Höhn; T. Grune (pp. 23-38).

Protein damage, caused by radicals, is involved in many diseases and in the aging process. Therefore, it is crucial to understand how protein damage can be limited, repaired or removed. To degrade damaged proteins, several intracellular proteolytic systems exist. One of the most important contributors in intracellular protein degradation of oxidized, aggregated and misfolded proteins is the proteasomal system. The proteasome is not a simple, unregulated structure. It is a more complex proteolytic composition that undergoes diverse regulation in situations of oxidative stress, aging and pathology. In addition to that, numerous studies revealed that the proteasome activity is altered during life time, contributing to the aging process. In addition, in the nervous system, the proteasome plays an important role in maintaining neuronal protein homeostasis. However, alterations in the activity may have an impact on the onset of neurodegenerative diseases. In this review, we discuss what is presently known about protein damage, the role of the proteasome in the degradation of damaged proteins and how the proteasome is regulated. Special emphasis was laid on the role of the proteasome in neurodegenerative diseases.

Keywords: Protein oxidation; Protein degradation; Proteasome; Neurodegeneration

Why do proteins use selenocysteine instead of cysteine? by Thomas Nauser; Daniel Steinmann; Willem H. Koppenol (pp. 39-44).

Selenocysteine is present in a variety of proteins and catalyzes the oxidation of thiols to disulfides and the reduction of disulfides to thiols. Here, we compare the kinetic and thermodynamic properties of cysteine with its selenium-containing analogon, selenocysteine. Reactions of simple selenols at pH 7 are up to four orders of magnitude faster than their sulfur analogs, depending on reaction type. In redox-related proteins, the use of selenium instead of sulfur can be used to tune electrode, or redox, potentials. Selenocysteine could also have a protective effect in proteins because its one-electron oxidized product, the selanyl radical, is not oxidizing enough to modify or destroy proteins, whereas the cysteine-thiyl radical can do this very rapidly.

Keywords: Kinetics; Thermodynamics; Sulfur; Selenium; Radical damage; Redox

Analytical methods for 3-nitrotyrosine quantification in biological samples: the unique role of tandem mass spectrometry by Dimitrios Tsikas (pp. 45-63).

Reactive-nitrogen species, such as peroxynitrite (ONOO⁻) and nitryl chloride (NO₂Cl), react with the aromatic ring of tyrosine in soluble amino acids and in proteins to form 3-nitrotyrosine. The extent of nitration can be quantified by measuring 3-nitrotyrosine in biological matrices, such as blood, urine, and tissue. This article reviews and discusses current analytical methodologies for the quantitative determination of 3-nitrotyrosine in their soluble and protein-associated forms, with the special focus being on free 3-nitrotyrosine. Special emphasis is given to analytical approaches based on the tandem mass spectrometry methodology. Pitfalls and solutions to overcome current methodological problems are emphasized and requirements for quantitative analytical approaches are recommended. The reliability of current analytical methods and the suitability of 3-nitrotyrosine as a biomarker of nitrative stress are thoroughly examined.

Keywords: Misidentification; Nitration; Peroxynitrite; Quantification; Tandem mass spectrometry; Tyrosine; Validation

Functional consequences of actin nitration: in vitro and in disease states by Mutay Aslan (pp. 65-74).

To link the phenomena of inflammatory-induced increases in protein nitrotyrosine (NO₂Tyr) derivatives to protein dysfunction and consequent pathological conditions, the evaluation of discrete NO₂Tyr modifications on specific proteins must be undertaken. Mass spectrometric (MS) proteomics-based strategies allow for the identification of all individual proteins that are nitrated by separating tissue homogenates using 2D gel electrophoresis, detecting the nitrated proteins using an anti-NO₂Tyr antibody, and then identifying the peptides generated during an in-gel proteolytic digest using matrix-assisted laser desorption ionization/time-of-flight (MALDI-TOF) MS. Actin, one of the most abundant proteins in eukaryotic cells, constitutes 5% or more of cell protein and serves with other cytoskeletal proteins as a critical target for nitration-induced functional impairment. Herein, examples of actin nitration detected under physiological conditions in various models of human disease or in clinically derived tissues are given and the impact that this post-translational protein modification can have on cell and organ function is discussed.

Keywords: Nitrotyrosine; Actin; Reactive nitrogen species; Reactive oxygen species; Protein structure and function

Reactive nitroxidative species and nociceptive processing: determining the roles for nitric oxide, superoxide, and peroxynitrite in pain by Joshua W. Little; Timothy Doyle; Daniela Salvemini (pp. 75-94).

Pain is a multidimensional perception and is modified at distinct regions of the neuroaxis. During enhanced pain, neuroplastic changes occur in the spinal and supraspinal nociceptive modulating centers and may result in a hypersensitive state termed central sensitization, which is thought to contribute to chronic pain states. Central sensitization culminates in hyperexcitability of dorsal horn nociceptive neurons resulting in increased nociceptive transmission and pain perception. This state is associated with enhanced nociceptive signaling, spinal glutamate-mediated N-methyl-d-aspartate receptor activation, neuroimmune activation, nitroxidative stress, and supraspinal descending facilitation. The nitroxidative species considered for their role in nociception and central sensitization include nitric oxide (NO), superoxide ( $${ ext {O}_2}^{{cdot }^{-}}$$ ), and peroxynitrite (ONOO⁻). Nitroxidative species are implicated during persistent but not normal nociceptive processing. This review examines the role of nitroxidative species in pain through a discussion of their contributions to central sensitization and the underlying mechanisms. Future directions for nitroxidative pain research are also addressed. As more selective pharmacologic agents are developed to target nitroxidative species, the exact role of nitroxidative species in pain states will be better characterized and should offer promising alternatives to available pain management options.

Keywords: Nitroxidative species; Peroxynitrite; Pain; Rostral ventromedial medulla (RVM); Superoxide dismutase mimetics; Peroxynitrite decomposition catalysts; Central sensitization; Neuroimmune activation

Design of Mn porphyrins for treating oxidative stress injuries and their redox-based regulation of cellular transcriptional activities by Ines Batinic-Haberle; Ivan Spasojevic; Hubert M. Tse; Artak Tovmasyan; Zrinka Rajic; Daret K. St. Clair; Zeljko Vujaskovic; Mark W. Dewhirst; Jon D. Piganelli (pp. 95-113).

The most efficacious Mn(III) porphyrinic (MnPs) scavengers of reactive species have positive charges close to the Mn site, whereby they afford thermodynamic and electrostatic facilitation for the reaction with negatively charged species such as O ₂ ^•− and ONOO⁻. Those are Mn(III) meso tetrakis(N-alkylpyridinium-2-yl)porphyrins, more specifically MnTE-2-PyP⁵⁺ (AEOL10113) and MnTnHex-2-PyP⁵⁺ (where alkyls are ethyl and n-hexyl, respectively), and their imidazolium analog, MnTDE-2-ImP⁵⁺ (AEOL10150, Mn(III) meso tetrakis(N,N′-diethylimidazolium-2-yl) porphyrin). The efficacy of MnPs in vivo is determined not only by the compound antioxidant potency, but also by its bioavailability. The former is greatly affected by the lipophilicity, size, structure, and overall shape of the compound. These porphyrins have the ability to both eliminate reactive oxygen species and impact the progression of oxidative stress-dependent signaling events. This will effectively lead to the regulation of redox-dependent transcription factors and the suppression of secondary inflammatory- and oxidative stress-mediated immune responses. We have reported on the inhibition of major transcription factors HIF-1α, AP-1, SP-1, and NF-κB by Mn porphyrins. While the prevailing mechanistic view of the suppression of transcription factors activation is via antioxidative action (presumably in cytosol), the pro-oxidative action of MnPs in suppressing NF-κB activation in nucleus has been substantiated. The magnitude of the effect is dependent upon the electrostatic (porphyrin charges) and thermodynamic factors (porphyrin redox ability). The pro-oxidative action of MnPs has been suggested to contribute at least in part to the in vitro anticancer action of MnTE-2-PyP⁵⁺ in the presence of ascorbate, and in vivo when combined with chemotherapy of lymphoma. Given the remarkable therapeutic potential of metalloporphyrins, future studies are warranted to further our understanding of in vivo action/s of Mn porphyrins, particularly with respect to their subcellular distribution.

Keywords: SOD mimics; Peroxynitrite scavengers; Mn porphyrins; Cellular transcriptional activity; MnTE-2-PyP⁵⁺ ; MnTnHex-2-PyP⁵⁺ ; NF-κB

Erratum to: Design of Mn porphyrins for treating oxidative stress injuries and their redox-based regulation of cellular transcriptional activities by Ines Batinic-Haberle; Ivan Spasojevic; Hubert M. Tse; Artak Tovmasyan; Zrinka Rajic; Daret K. St. Clair; Zeljko Vujaskovic; Mark W. Dewhirst; Jon D. Piganelli (pp. 115-116).

Protein damage by photo-activated Zn(II) N-alkylpyridylporphyrins by Ludmil Benov; James Craik; Ines Batinic-Haberle (pp. 117-128).

Destruction of unwanted cells and tissues in photodynamic therapy (PDT) is achieved by a combination of light, oxygen, and light-sensitive molecules. The advantages of PDT compared to other traditional treatment modalities, and the shortcomings of the currently used photosensitizers, have stimulated the search for new, more efficient photosensitizer candidates. Ability to inflict selective damage to particular proteins through photo-irradiation would significantly advance the design of highly specific photosensitizers. Achieving this objective requires comprehensive knowledge concerning the interactions of the particular photosensitizer with specific targets. Here, we summarize the effects of Zn(II) N-alkylpyridylporphyrin-based photosensitizers on intracellular (metabolic, antioxidant and mitochondrial enzymes) and membrane proteins. We emphasize how the structural modifications of the porphyrin side substituents affect their lipophilicity, which in turn influence their subcellular localization. Thus, Zn(II) N-alkylpyridylporphyrins target particular cellular sites and proteins of interest, and are more efficient than hematoporphyrin D, whose commercial preparation (Photofrin) has been clinically approved for PDT.

Keywords: Ortho, meta, and para isomeric Zn(II) N-alkylpyridyl porphyrins; Photosensitizer; Hematoporphyrin D (HpD); Oxidative protein modification; Photodynamic therapy

Existing bioinformatics tools for the quantitation of post-translational modifications by Jens Allmer (pp. 129-138).

Mass spectrometry (MS)-based proteomics, by itself, is a vast and complex area encompassing various mass spectrometers, different spectra, and search result representations. When the aim is quantitation performed in different scanning modes at different MS levels, matters become additionally complex. Quantitation of post-translational modifications (PTM) represents the greatest challenge among these endeavors. Many different approaches to quantitation have been described and some of these can be directly applied to the quantitation of PTMs. The amount of data produced via MS, however, makes manual data interpretation impractical. Therefore, specialized software tools meet this challenge. Any software currently able to quantitate differentially labeled samples may theoretically be adapted to quantitate differential PTM expression among samples as well. Due to the heterogeneity of mass spectrometry-based proteomics; this review will focus on quantitation of PTM using liquid chromatography followed by one or more stages of mass spectrometry. Currently available free software, which either allow analysis of PTM or are easily adaptable for this purpose, is briefly reviewed in this paper. Selected studies, especially those related to phosphoproteomics, shall be used to highlight the current ability to quantitate PTMs.

Keywords: Quantitation; Quantification; Post-translational modification; Software; LC-MS; PTM

Manganese superoxide dismutase: beyond life and death by Aaron K. Holley; Sanjit Kumar Dhar; Yong Xu; Daret K. St. Clair (pp. 139-158).

Manganese superoxide dismutase (MnSOD) is a nuclear-encoded antioxidant enzyme that localizes to the mitochondria. Expression of MnSOD is essential for the survival of aerobic life. Transgenic mice expressing a luciferase reporter gene under the control of the human MnSOD promoter demonstrate that the level of MnSOD is reduced prior to the formation of cancer. Overexpression of MnSOD in transgenic mice reduces the incidences and multiplicity of papillomas in a DMBA/TPA skin carcinogenesis model. However, MnSOD deficiency does not lead to enhanced tumorigenicity of skin tissue similarly treated because MnSOD can modulate both the p53-mediated apoptosis and AP-1-mediated cell proliferation pathways. Apoptosis is associated with an increase in mitochondrial levels of p53 suggesting a link between MnSOD deficiency and mitochondrial-mediated apoptosis. Activation of p53 is preventable by application of a SOD mimetic (MnTE-2-PyP⁵⁺). Thus, p53 translocation to mitochondria and subsequent inactivation of MnSOD explain the observed mitochondrial dysfunction that leads to transcription-dependent mechanisms of p53-induced apoptosis. Administration of MnTE-2-PyP⁵⁺ following apoptosis but prior to proliferation leads to suppression of protein carbonyls and reduces the activity of AP-1 and the level of the proliferating cellular nuclear antigen, without reducing the activity of p53 or DNA fragmentation following TPA treatment. Remarkably, the incidence and multiplicity of skin tumors are drastically reduced in mice that receive MnTE-2-PyP⁵⁺ prior to cell proliferation. The results demonstrate the role of MnSOD beyond its essential role for survival and suggest a novel strategy for an antioxidant approach to cancer intervention.

Keywords: MnSOD; p53; Cancer; Chemotherapy

Maintenance of antioxidant defenses of brain cells: plasma membrane glutamate transporters and beyond by Laurence Had-Aissouni (pp. 159-161).

Modulation of neuronal glutathione synthesis by EAAC1 and its interacting protein GTRAP3-18 by Koji Aoyama; Masahiko Watabe; Toshio Nakaki (pp. 163-169).

Glutathione (GSH) plays essential roles in different processes such as antioxidant defenses, cell signaling, cell proliferation, and apoptosis in the central nervous system. GSH is a tripeptide composed of glutamate, cysteine, and glycine. The concentration of cysteine in neurons is much lower than that of glutamate or glycine, so that cysteine is the rate-limiting substrate for neuronal GSH synthesis. Most neuronal cysteine uptake is mediated through the neuronal sodium-dependent glutamate transporter, known as excitatory amino acid carrier 1 (EAAC1). Glutamate transporters are vulnerable to oxidative stress and EAAC1 dysfunction impairs neuronal GSH synthesis by reducing cysteine uptake. This may start a vicious circle leading to neurodegeneration. Intracellular signaling molecules functionally regulate EAAC1. Glutamate transporter-associated protein 3-18 (GTRAP3-18) activation down-regulates EAAC1 function. Here, we focused on the interaction between EAAC1 and GTRAP3-18 at the plasma membrane to investigate their effects on neuronal GSH synthesis. Increased level of GTRAP3-18 protein induced a decrease in GSH level and, thereby, increased the vulnerability to oxidative stress, while decreased level of GTRAP3-18 protein induced an increase in GSH level in vitro. We also confirmed these results in vivo. Our studies demonstrate that GTRAP3-18 regulates neuronal GSH level by controlling the EAAC1-mediated uptake of cysteine.

Keywords: Glutathione; Cysteine; EAAC1; GTRAP3-18; Neurodegeneration

Regulation of xCT expression and system $$ x_{ ext{c}}^{ - } $$ function in neuronal cells by Jan Lewerenz; Pamela Maher; Axel Methner (pp. 171-179).

The glutamate/cystine antiporter system $$ x_{ ext{c}}^{ - } $$ transports cystine into cells in exchange for glutamate at a ratio of 1:1. It is composed of a specific light chain, xCT, and a heavy chain, 4F2, linked by a disulfide bridge. Intracellularly, cystine is reduced into cysteine, the rate-limiting precursor of glutathione (GSH), an important small molecule antioxidant. Several lines of evidence suggest that the expression of xCT and thereby the presence system $$ x_{ ext{c}}^{ - } $$ activity plays an important role in the brain. First, it regulates extracellular glutamate concentrations. Second, as brain is prone to oxidative stress due to its high oxygen consumption and lipid content, system $$ x_{ ext{c}}^{ - } , $$ by favoring GSH synthesis, may prevent oxidative damage. Thus, to understand how xCT expression and system $$ x_{ ext{c}}^{ - } $$ activity are regulated in the central nervous system is of utmost importance. In this review, we will summarize the current knowledge about the molecular basis by which xCT expression and system $$ x_{ ext{c}}^{ - } $$ activity are regulated in neuronal cell lines, especially the hippocampal cell line, HT22. In addition, we will relate these pathways to findings in other cell types, especially those found in the central nervous system. We will focus on the signaling pathways that modulate the transcription of the xCT gene. Furthermore, we describe possible pathways that modify system $$ x_{ ext{c}}^{ - } $$ activity beyond the level of xCT transcription, including regulation on the level of membrane trafficking and substrate availability, especially the regulation by glutamate transport through excitatory amino acid transporters.

Keywords: Cystine; Glutamate; xCT; Oxidative stress; Neurons

Toward a new role for plasma membrane sodium-dependent glutamate transporters of astrocytes: maintenance of antioxidant defenses beyond extracellular glutamate clearance by Laurence Had-Aissouni (pp. 181-197).

The primary function assigned to the sodium-dependent glutamate transporters, also known as excitatory amino acid transporters (EAATs), is to maintain the extracellular glutamate concentration in the low micromolar range, allowing glutamate to be used as a signaling molecule in the brain and preventing its cytotoxic effects. However, glutamate and cyst(e)ine, that is also a substrate of EAATs, are also important metabolites used for instance in the synthesis of the main antioxidant glutathione. This review describes the evidence suggesting that EAATs, by providing glutathione precursors, are crucial to prevent oxidative death in particular cells of the nervous system while being dispensable in others. This differential importance may depend on the way antioxidant defenses are maintained in each cell type and on the metabolic fate of transported substrates, both being probably controlled by EAAT interacting proteins. As oxidative stress invariably contributes to various forms of cell death, a better understanding of how antioxidant defenses are maintained in particular brain cells will probably help to develop protective strategies in degenerative insults specifically affecting these cells.

Keywords: Astrocytes; EAATs; Glutathione; Oxidative stress; Ischemia; Parkinson’s disease

The transsulfuration pathway: a source of cysteine for glutathione in astrocytes by Gethin J. McBean (pp. 199-205).

Astrocyte cells require cysteine as a substrate for glutamate cysteine ligase (γ-glutamylcysteine synthase; EC 6.3.2.2) catalyst of the rate-limiting step of the γ-glutamylcycle leading to formation of glutathione (l-γ-glutamyl-l-cysteinyl-glycine; GSH). In both astrocytes and glioblastoma/astrocytoma cells, the majority of cysteine originates from reduction of cystine imported by the x _c ⁻ cystine-glutamate exchanger. However, the transsulfuration pathway, which supplies cysteine from the indispensable amino acid, methionine, has recently been identified as a significant contributor to GSH synthesis in astrocytes. The purpose of this review is to evaluate the importance of the transsulfuration pathway in these cells, particularly in the context of a reserve pathway that channels methionine towards cysteine when the demand for glutathione is high, or under conditions in which the supply of cystine by the x _c ⁻ exchanger may be compromised.

Keywords: Transsulfuration; Brain; Astrocyte; Cysteine; Cystathionine-γ-lyase; Cystathionine-β-synthase; Glutathione

Microglial self-defence mediated through GLT-1 and glutathione by Mikael Persson; Lars Rönnbäck (pp. 207-219).

Glutamate is stored in synaptic vesicles in presynaptic neurons. It is released into the synaptic cleft to provide signalling to postsynaptic neurons. Normally, the astroglial glutamate transporters GLT-1 and GLAST take up glutamate to mediate a high signal-to-noise ratio in the synaptic signalling, and also to prevent excitotoxic effects by glutamate. In astrocytes, glutamate is transformed into glutamine, which is safely transported back to neurons. However, in pathological conditions, such as an ischemia or virus infection, astroglial transporters are down-regulated which could lead to excitotoxicity. Lately, it was shown that even microglia can express glutamate transporters during pathological events. Microglia have two systems for glutamate transport: GLT-1 for transport into the cells and the x _c ⁻ system for transport out of the cells. We here review results from our work and others, which demonstrate that microglia in culture express GLT-1, but not GLAST, and transport glutamate from the extracellular space. We also show that TNF-α can induce increased microglial GLT-1 expression, possibly associating the expression with inflammatory systems. Furthermore, glutamate taken up through GLT-1 may be used for direct incorporation into glutathione and to fuel the intracellular glutamate pool to allow cystine uptake through the x _c ⁻ system. This can lead to a defence against oxidative stress and have an antiviral function.

Keywords: Microglia; Glutamate; GLT-1; Glutathione; Neuroinflammation

EAAT expression by macrophages and microglia: still more questions than answers by Gabriel Gras; Boubekeur Samah; Audrey Hubert; Cathie Léone; Fabrice Porcheray; Anne-Cécile Rimaniol (pp. 221-229).

Glutamate is the main excitatory amino acid, but its presence in the extracellular milieu has deleterious consequences. It may induce excitotoxicity and also compete with cystine for the use of the cystine–glutamate exchanger, blocking glutathione neosynthesis and inducing an oxidative stress-induced cell death. Both mechanisms are critical in the brain where up to 20% of total body oxygen consumption occurs. In normal conditions, the astrocytes ensure that extracellular concentration of glutamate is kept in the micromolar range, thanks to their coexpression of high-affinity glutamate transporters (EAATs) and glutamine synthetase (GS). Their protective function is nevertheless sensitive to situations such as oxidative stress or inflammatory processes. On the other hand, macrophages and microglia do not express EAATs and GS in physiological conditions and are the principal effector cells of brain inflammation. Since the late 1990s, a number of studies have now shown that both microglia and macrophages display inducible EAAT and GS expression, but the precise significance of this still remains poorly understood. Brain macrophages and microglia are sister cells but yet display differences. Both are highly sensitive to their microenvironment and can perform a variety of functions that may oppose each other. However, in the very particular environment of the healthy brain, they are maintained in a repressed state. The aim of this review is to present the current state of knowledge on brain macrophages and microglial cells activation, in order to help clarify their role in the regulation of glutamate under pathological conditions as well as its outcome.

Keywords: Microglia; Macrophage; EAAT; xCT; Glutamate; Glutathione; Neuroinflammation

The oxidative stress-inducible cystine/glutamate antiporter, system x _c ⁻ : cystine supplier and beyond by Marcus Conrad; Hideyo Sato (pp. 231-246).

The oxidative stress-inducible cystine/glutamate exchange system, system x _c ⁻ , transports one molecule of cystine, the oxidized form of cysteine, into cells and thereby releases one molecule of glutamate into the extracellular space. It consists of two protein components, the 4F2 heavy chain, necessary for membrane location of the heterodimer, and the xCT protein, responsible for transport activity. Previously, system x _c ⁻ has been regarded to be a mere supplier of cysteine to cells for the synthesis of proteins and the antioxidant glutathione (GSH). In that sense, oxygen, electrophilic agents, and bacterial lipopolysaccharide trigger xCT expression to accommodate with increased oxidative stress by stimulating GSH biosynthesis. However, emerging evidence established that system x _c ⁻ may act on its own as a GSH-independent redox system by sustaining a redox cycle over the plasma membrane. Hallmarks of this cycle are cystine uptake, intracellular reduction to cysteine and secretion of the surplus of cysteine into the extracellular space. Consequently, increased levels of extracellular cysteine provide a reducing microenvironment required for proper cell signaling and communication, e.g. as already shown for the mechanism of T cell activation. By contrast, the enhanced release of glutamate in exchange with cystine may trigger neurodegeneration due to glutamate-induced cytotoxic processes. This review aims to provide a comprehensive picture from the early days of system x _c ⁻ research up to now.

Keywords: 4F2; Cystine/cysteine redox cycle; Glutamate; Glutathione; Slc7a11 ; xCT

Fast analysis of wine for total homocysteine content by high-performance liquid chromatography by Rafał Głowacki; Kamila Borowczyk; Edward Bald (pp. 247-251).

Alimentary methionine is believed to be the main source for plasma homocysteine. Recent literature supplies information about homocysteine content in daily food components, but not in wine, an attractive complement of the evening meal in some western countries. In this communication, a simple and fast high-performance liquid chromatography method for determination of total homocysteine in wine is described. The two steps procedure relies on reduction of the disulfide forms of homocysteine with tris-(2-carboxyethyl)phosphine and on-column derivatization with o-phthaldialdehyde followed by separation and fluorescence detection. The entire analysis time, including sample work-up, amounts 14 min. The calibration performed with wine matrix, spiked with homocystine within the practical concentration range, proved linear response of the detector. The proposed method was applied for the analysis of 32 different types of wines for total homocysteine. The average concentration of the analyte was 10.31 (±4.25) µM and 6.11 (±3.44) µM for red (n = 23) and white (n = 9) wines, respectively.

Keywords: Homocysteine; Wine; Determination; Liquid chromatography

Comparative study of the interaction of synthetic methionine-enkephalin and its amidated derivate with monolayers of zwitterionic and negatively charged phospholipids by Asya Tsanova; A. Jordanova; G. As. Georgiev; T. Pajpanova; E. Golovinsky; Z. Lalchev (pp. 253-260).

Using Langmuir’s monolayer technique, the surface behavior and the interaction of the synthetic neuropeptide methionine-enkephalin (Met-enk) and its amidated derivate (Met-enk-NH₂) with monolayers of the zwitterionic dimyristoylphosphatidylcholine (DMPC) and the negatively charged dimyristoylphosphatidylglycerol (DMPG) were studied. The surface tension (γ, mN/m) of DMPG and DMPC monolayers as a function of time (after injection of the peptide under the interface) was detected. The decrease in γ values showed that there was a strong penetration effect of both types of Met-enk molecules into the monolayers, being significantly stronger for the amidated derivate, Met-enk-NH₂. We suggest that the interaction between the neuropeptides and DMPC was predominantly determined by peptides amphiphilicity, while the electrostatic forces play significant role for the insertion of the cationic Met-enk-NH₂ in DMPG monolayers, especially at high packing densities. Our results demonstrate the potential of lipid monolayers formed in Langmuir’s trough to be successfully used as an elegant and simple membrane models to study lipid–peptide interactions at the air/water interface.

Keywords: Methionine-enkephalin; DMPC; DMPG; Langmuir’s monolayer technique; Surface tension

CNS delivery of l-dopa by a new hybrid glutathione–methionine peptidomimetic prodrug by Francesco Pinnen; Ivana Cacciatore; Catia Cornacchia; Adriano Mollica; Piera Sozio; Laura S. Cerasa; Antonio Iannitelli; Antonella Fontana; Cinzia Nasuti; Antonio Di Stefano (pp. 261-269).

Parkinson’s disease (PD) is a neurodegenerative disorder associated primarily with loss of dopamine (DA) neurons in the nigrostriatal system. With the aim of increasing the bioavailability of l-dopa (LD) after oral administration and of overcoming the pro-oxidant effect associated with LD therapy, we designed a peptidomimetic LD prodrug (1) able to release the active agent by enzyme catalyzed hydrolysis. The physicochemical properties, as well as the chemical and enzymatic stabilities of the new compound, were evaluated in order to check both its stability in aqueous medium and its sensitivity towards enzymatic cleavage, providing the parent LD drug, in rat and human plasma. The radical scavenging activities of prodrug 1 was tested by using both the DPPH–HPLC and the DMSO competition methods. The results indicate that the replacement of cysteine GSH portion by methionine confers resistance to oxidative degradation in gastric fluid. Prodrug 1 demonstrated to induce sustained delivery of DA in rat striatal tissue with respect to equimolar LD dosages. These results are of significance for prospective therapeutic application of prodrug 1 in pathological events associated with free radical damage and decreasing DA concentration in the brain.

Keywords: l-Dopa; Glutathione; Methionine; Parkinson’s disease

Determination of protein folding kinetic types using sequence and predicted secondary structure and solvent accessibility by Hua Zhang; Tuo Zhang; Jianzhao Gao; Jishou Ruan; Shiyi Shen; Lukasz Kurgan (pp. 271-283).

Proteins fold through a two-state (TS), with no visible intermediates, or a multi-state (MS), via at least one intermediate, process. We analyze sequence-derived factors that determine folding types by introducing a novel sequence-based folding type predictor called FOKIT. This method implements a logistic regression model with six input features which hybridize information concerning amino acid composition and predicted secondary structure and solvent accessibility. FOKIT provides predictions with average Matthews correlation coefficient (MCC) between 0.58 and 0.91 measured using out-of-sample tests on four benchmark datasets. These results are shown to be competitive or better than results of four modern predictors. We also show that FOKIT outperforms these methods when predicting chains that share low similarity with the chains used to build the model, which is an important advantage given the limited number of annotated chains. We demonstrate that inclusion of solvent accessibility helps in discrimination of the folding kinetic types and that three of the features constitute statistically significant markers that differentiate TS and MS folders. We found that the increased content of exposed Trp and buried Leu are indicative of the MS folding, which implies that the exposure/burial of certain hydrophobic residues may play important role in the formation of the folding intermediates. Our conclusions are supported by two case studies.

Keywords: Folding; Folding kinetic types; Folding rate; Solvent accessibility; Secondary structure

A practical synthesis of N α-Fmoc protected l-threo-β-hydroxyaspartic acid derivatives for coupling via α- or β-carboxylic group by Nina Bionda; Maré Cudic; Lidija Barisic; Maciej Stawikowski; Roma Stawikowska; Diego Binetti; Predrag Cudic (pp. 285-293).

A simple and practical general synthetic protocol towards orthogonally protected tHyAsp derivatives fully compatible with Fmoc solid-phase peptide synthetic methodology is reported. Our approach includes enantioresolution of commercially available d,l-tHyAsp racemic mixture by co-crystallization with l-Lys, followed by ion exchange chromatography yielding enantiomerically pure l-tHyAsp and d-tHyAsp, and their selective orthogonal protection. In this way N α-Fmoc protected tHyAsp derivatives were prepared ready for couplings via either α- or β-carboxylic group onto the resins or the growing peptide chain. In addition, coupling of tHyAsp via β-carboxylic group onto amino resins allows preparation of peptides containing tHyAsn sequences, further increasing the synthetic utility of prepared tHyAsp derivatives.

Keywords: Hydroxyaspartic acid; Enantioresolution; Orthogonal protection; Fmoc solid-phase peptide synthesis

Living with high putrescine: expression of ornithine and arginine biosynthetic pathway genes in high and low putrescine producing poplar cells by Andrew F. Page; Rakesh Minocha; Subhash C. Minocha (pp. 295-308).

Arginine (Arg) and ornithine (Orn), both derived from glutamate (Glu), are the primary substrates for polyamine (PA) biosynthesis, and also play important roles as substrates and intermediates of overall N metabolism in plants. Their cellular homeostasis is subject to multiple levels of regulation. Using reverse transcription quantitative PCR (RT-qPCR), we studied changes in the expression of all genes of the Orn/Arg biosynthetic pathway in response to up-regulation [via transgenic expression of mouse Orn decarboxylase (mODC)] of PA biosynthesis in poplar (Populus nigra × maximowiczii) cells grown in culture. Cloning and sequencing of poplar genes involved in the Orn/Arg biosynthetic pathway showed that they have high homology with similar genes in other plants. The expression of the genes of Orn, Arg and PA biosynthetic pathway fell into two hierarchical clusters; expression of one did not change in response to high putrescine, while members of the other cluster showed a shift in expression pattern during the 7-day culture cycle. Gene expression of branch point enzymes (N-acetyl-Glu synthase, Orn aminotransferase, Arg decarboxylase, and spermidine synthase) in the sub-pathways, constituted a separate cluster from those involved in intermediary reactions of the pathway (N-acetyl-Glu kinase, N-acetyl-Glu-5-P reductase, N-acetyl-Orn aminotransferase, N ₂-acetylOrn:N-acetyl-Glu acetyltransferase, N ₂-acetyl-Orn deacetylase, Orn transcarbamylase, argininosuccinate synthase, carbamoylphosphate synthetase, argininosuccinate lyase, S-adenosylmethionine decarboxylase, spermine synthase). We postulate that expression of all genes of the Glu-Orn-Arg pathway is constitutively coordinated and is not influenced by the increase in flux rate through this pathway in response to increased utilization of Orn by mODC; thus the pathway involves mostly biochemical regulation rather than changes in gene expression. We further suggest that Orn itself plays a major role in the regulation of this pathway.

Keywords: Arginine biosynthesis; Ornithine biosynthesis; Gene expression; Polyamines; Reverse transcriptase quantitative polymerase chain reaction; Hierarchical clustering

Synthesis of 4-thia-[6-¹³C]lysine from [2-¹³C]glycine: access to site-directed isotopomers of 2-aminoethanol, 2-bromoethylamine and 4-thialysine by Amarendra Nath Maity; Ajam C. Shaikh; Sankareswaran Srimurugan; Chi-Ju Wu; Chinpiao Chen; Shyue-Chu Ke (pp. 309-315).

4-Thialysine (S-(2-aminoethyl)-l-cysteine) is an analog of lysine. It has been used as an alternative substrate for lysine in enzymatic reactions. Site-directed isotopomers are often needed for elucidation of mechanism of reactions. 4-Thialysine can be synthesized by reacting cysteine with 2-bromoethylamine, an important reagent in chemical-modification rescue (CMR) of proteins. Here, we present the synthesis of 4-thia-[6-¹³C]lysine, one of the isotopomers of 4-thialysine, from commercially available starting material [2-¹³C]glycine via formation of five intermediates including 2-amino[2-¹³C]ethanol and 2-bromo[1-¹³C]ethylamine. The compounds were characterized using various spectroscopic techniques. Moreover, we discuss that our strategy would provide access to site-directed isotopomers of 2-aminoethanol, 2-bromoethylamine and 4-thialysine. Biological activity of 4-thia-[6-¹³C]lysine was tested in the enzymatic reaction of lysine 5,6-aminomutase.

Keywords: Amino acids; Carbon labels; Site-directed isotopomers; 4-Thialysine; 2-Bromoethylamine; 2-Aminoethanol

Differential responses of microsomal proteins and metabolites in two contrasting cadmium (Cd)-accumulating soybean cultivars under Cd stress by Nagib Ahsan; Takuji Nakamura; Setsuko Komatsu (pp. 317-327).

While there are significant genotypic differences in cadmium (Cd) uptake and distribution in soybean cultivars, little attention has been paid to the underlying molecular mechanisms. We adopted a comparative proteomic approach coupled with metabolite analysis to examine Cd uptake and translocation in two contrasting Cd-accumulating soybean cultivars, Enrei and Harosoy, which accumulate higher amount of Cd in the roots and aerial parts, respectively. Proteins extracted from the root microsomal fraction were evaluated by immunoblot analysis using different subcellular marker proteins. Analysis of control and Cd-exposed samples by two-dimensional gel electrophoresis coupled with mass spectrometry revealed a total of 13 and 11 differentially expressed proteins in the Enrei and Harosoy cultivars, respectively. Metabolome profiling identified a total of 32 metabolites, the expression of 18 of which was significantly altered in at least in one cultivar in response to Cd stress. Analysis of the combined proteomic and metabolomic results revealed that proteins and amino acids associate with Cd-chelating pathways are highly active in the Enrei cultivar. In addition, proteins associated with lignin biosynthesis are significantly upregulated in the Enrei cultivar under Cd stress. Our results indicate that in the Enrei cultivar, Cd-chelating agents may bind excess free Cd ion and that translocation of Cd from the roots to the aerial parts might be prevented by increased xylem lignification.

Keywords: Cadmium; Lignifications; Metablomics; Proteomics; Soybean

A leucine-rich diet and exercise affect the biomechanical characteristics of the digital flexor tendon in rats after nutritional recovery by Alexandre Wesley Carvalho Barbosa; Gustavo Pereira Benevides; Leda Maria Totti Alferes; Emilianne Miguel Salomão; Maria Cristina Cintra Gomes-Marcondes; Laurecir Gomes (pp. 329-336).

An increase in the capacity of athletic performance depends on adequate nutrition, which ensures optimal function of the musculoskeletal system, including tendon stability. However, little is known about the status of tendons and extracellular matrix modifications during malnutrition and nutritional recovery when leucine is used in response to exercise conditioning. The purpose of this study was to evaluate the collagen content and biomechanical aspects of the deep digital flexor tendon (DDFT) in malnourished rats submitted to nutritional recovery (control diet or leucine-rich diet) and aerobic physical activity. After 60 days of undernourishment (6% protein diet), the malnourished rats were subsequently nutritionally recovered with a control diet or leucine-rich diet and trained or not (swimming, without overload) for 5 weeks. The biomechanical analysis and quantification of hydroxyproline were assessed in the DDFT in all experimental groups. The leucine-rich diet increased hydroxyproline content in the tension region, independently of the training. In the compression region, hydroxyproline content was higher in the malnourished and leucine-trained groups. Biomechanical analysis showed a lower load in the malnourished and all-trained groups. The lowest stress was observed with control-trained animals. The nutritional-recovered groups showed higher strain values corresponding to control group, while the lowest values were observed in malnourished and trained groups. The results suggest that a leucine-rich diet stimulates collagen synthesis of the DDFT, especially when in combination with physical exercise, and seems to determine the increase of resistance and the biomechanical characteristics of tendons.

Keywords: Leucine; Aerobic exercise; Extracellular matrix; Tendon; Nutrition

Study on the decrease of renal d-amino acid oxidase activity in the rat after renal ischemia by chiral ligand exchange capillary electrophoresis by Haizhi Zhang; Li Qi; Yuqing Lin; Lanqun Mao; Yi Chen (pp. 337-345).

d-Amino acid oxidase (DAAO) in mammal kidney regulates the renal reactive oxygen species (ROS) levels directly and plays a leading role in the development of ROS-mediated renal pathologic damages based on its crucial role in the oxidative deamination of d-amino acids and the consequent generation of H₂O₂. Quantitative measurement of DAAO activity in the process of renal ischemia, which could help to understand the molecular mechanisms of this gripping acute renal disease, was conducted through the determination of chiral substrate by capillary electrophoresis (CE) in our study. In this study, a chiral ligand exchange CE method was explored with Zn(II)-l-alaninamide complex as the chiral selector to investigate DAAO activity by determining the decreased concentration of the chiral substrate of DAAO-mediated enzymatic reaction. Then, the change of DAAO activity following 60-min acute renal ischemia in rats was observed with the proposed method. The study showed that the operation of renal ischemia resulted in a 45.49 ± 8.30% (n = 8) decrease in the DAAO-induced consumption of substrate, indicating a sharp decrease in renal DAAO activity following this acute renal injury. This phenomenon, with the possible reason of metabolic acidosis, could pave a new way for the study of oxidative stress in the development of renal ischemia injury.

Keywords: d-Amino acid oxidase (DAAO); Renal ischemia; Chiral ligand-exchange capillary electrophoresis; d,l-Methionine

Lysine transporters in human trypanosomatid pathogens by Ehud Inbar; Gaspar E. Canepa; Carolina Carrillo; Fabian Glaser; Marianne Suter Grotemeyer; Doris Rentsch; Dan Zilberstein; Claudio A. Pereira (pp. 347-360).

In previous studies we characterized arginine transporter genes from Trypanosoma cruzi and Leishmania donovani, the etiological agents of chagas disease and kala azar, respectively, both fatal diseases in humans. Unlike arginine transporters in higher eukaryotes that transport also lysine, these parasite transporters translocate only arginine. This phenomenon prompted us to identify and characterize parasite lysine transporters. Here we demonstrate that LdAAP7 and TcAAP7 encode lysine-specific permeases in L. donovani and T. cruzi, respectively. These two lysine permeases are both members of the large amino acid/auxin permease family and share certain biochemical properties, such as specificity and Km. However, we evidence that LdAAP7 and TcAAP7 differ in their regulation and localization, such differences are likely a reflection of the dissimilar L. donovani and T. cruzi life cycles. Failed attempts to delete both alleles of LdAAP7 support the premise that this is an essential gene that encodes the only lysine permeases expressed in L. donovani promastigotes and T. cruzi epimastigotes, respectively.

Keywords: Lysine transport; Amino acid transport; Leishmania ; Trypanosoma

Structural basis for the stability of a thermophilic methionine adenosyltransferase against guanidinium chloride by Francisco Garrido; John C. Taylor; Carlos Alfonso; George D. Markham; María A. Pajares (pp. 361-373).

The methionine adenosyltransferase from the thermophile Methanococcus jannaschii is fully and irreversibly unfolded in the presence of guanidinium chloride. Unfolding of this dimeric protein is a three-state process in which a dimeric intermediate could be identified. The less stable secondary structural elements of the protein are the C-terminal ends of β-strands E2 and E6, as deduced from the behavior of tyrosine to tryptophan mutants at residues 72 and 170, which are located in the subunit interface. Unraveling of these elements at the monomer interface may soften intersubunit interactions, leading to the observed 85% activity loss. Accumulation of the intermediate was associated with maintenance of residual activity, an increase in the elution volume of the protein upon gel filtration and a decrease in the sedimentation coefficient. Elimination of the remaining enzymatic activity occurred in conjunction with a 50% reduction in helicity and fluorescence alterations illustrating a transient burial of tryptophans at β-strands E2, E3 and E9. The available 3D-model predicted that these β-strands are involved in the central and N-terminal domains of the monomer structure. Severe perturbation of this area of the monomer–monomer interface may destroy the remaining intermolecular interactions, thus leading to dissociation and aggregation. Finally, transition to the denatured state includes completion of the changes detected in the microenvironments around tryptophans included at α-helixes H5 and H6, the loops connecting H5–E8 and E9, β-strands E3 and E12.

Keywords: Methionine adenosyltransferase; Unfolding; Stability; Guanidinium chloride; Thermophile

Effects of essential amino acids or glutamine deprivation on intestinal permeability and protein synthesis in HCT-8 cells: involvement of GCN2 and mTOR pathways by Nabile Boukhettala; Sophie Claeyssens; Malik Bensifi; Brigitte Maurer; Juliette Abed; Alain Lavoinne; Pierre Déchelotte; Moïse Coëffier (pp. 375-383).

GCN2 and mTOR pathways are involved in the regulation of protein metabolism in response to amino acid availability in different tissues. However, regulation at intestinal level is poorly documented. The aim of the study was to evaluate the effects of a deprivation of essential amino acids (EAA) or glutamine (Gln) on these pathways in intestinal epithelial cells. Intestinal epithelial cell, HCT-8, were incubated during 6 h with 1/DMEM culture medium containing EAA, non EAA and Gln, 2/with saline as positive control of nutritional deprivation, 3/DMEM without EAA, 4/DMEM without Gln or 5/DMEM without Gln and supplemented with a glutamine synthase inhibitor (MSO, 4 mM). Intestinal permeability was evaluated by the measure of transepithelial electric resistance (TEER). Using [L-²H₃]-leucine incorporation, fractional synthesis rate (FSR) was calculated from the assessed enrichment in proteins and free amino acid pool by GCMS. Expression of eiF2α (phosphorylated or not), used as marker of GCN2 pathway, and of 4E-BP1 (phosphorylated or not), used as a marker of mTOR pathway, was evaluated by immunoblot. Results were compared by ANOVA. Six-hours EAA deprivation did not significantly affect TEER and FSR but decreased p-4E-BP1 and increased p-eiF2α. In contrast, Gln deprivation decreased FSR and p-4E-BP1. MSO induced a marked decrease of TEER and FSR and an increase of p-eiF2α, whereas mTOR pathway remained activated. These results suggest that both mTOR and GCN2 pathways can mediate the limiting effects of Gln deprivation on protein synthesis according to its severity.

Keywords: Amino acids; Cellular signaling; Glutamine; Intestine; Nutrition

Antitumor and angiostatic peptides from frog skin secretions by Hanneke van Zoggel; Yamina Hamma-Kourbali; Cécile Galanth; Ali Ladram; Pierre Nicolas; José Courty; Mohamed Amiche; Jean Delbé (pp. 385-395).

The discovery of new molecules with potential antitumor activity continues to be of great importance in cancer research. In this respect, natural antimicrobial peptides isolated from various animal species including humans and amphibians have been found to be of particular interest. Here, we report the presence of two anti-proliferative peptides active against cancer cells in the skin secretions of the South American tree frog, Phyllomedusa bicolor. The crude skin exudate was fractioned by size exclusion gel followed by reverse-phase HPLC chromatography. After these two purification steps, we identified two fractions that exhibited anti-proliferative activity. Sequence analysis indicated that this activity was due to two antimicrobial α-helical cationic peptides of the dermaseptin family (dermaseptins B2 and B3). This result was confirmed using synthetic dermaseptins. When tested in vitro, synthetic B2 and B3 dermaseptins inhibited the proliferation of the human prostatic adenocarcinoma PC-3 cell line by more than 90%, with an EC₅₀ of around 2–3 μM. No effect was observed on the growth of the NIH-3T3 non-tumor mouse cell line with Drs B2, whereas a slight inhibiting effect was observed with Drs B3 at high dose. In addition, the two fractions obtained after size exclusion chromatography also inhibited PC-3 cell colony formation in soft agar. Interestingly, inhibition of the proliferation and differentiation of activated adult bovine aortic endothelial cells was observed in cells treated with these two fractions. Dermaseptins B2 and B3 could, therefore, represent interesting new pharmacological molecules with antitumor and angiostatic properties for the development of a new class of anticancer drugs.

Keywords: Antimicrobial peptide; Dermaseptin; Antitumor activity; Angiogenesis

Sections

Personal tools

Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.42, #1)

Sections

Personal tools

Local resources

Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.42, #1)