Amino Acids (v.25, #3-4)

 We summarize here results of studies designed to elucidate basic mechanisms of reactive oxygen (ROS)-mediated oxidation of proteins and free amino acids. These studies have shown that oxidation of proteins can lead to hydroxylation of aromatic groups and aliphatic amino acid side chains, nitration of aromatic amino acid residues, nitrosylation of sulfhydryl groups, sulfoxidation of methionine residues, chlorination of aromatic groups and primary amino groups, and to conversion of some amino acid residues to carbonyl derivatives. Oxidation can lead also to cleavage of the polypeptide chain and to formation of cross-linked protein aggregates. Furthermore, functional groups of proteins can react with oxidation products of polyunsaturated fatty acids and with carbohydrate derivatives (glycation/glycoxidation) to produce inactive derivatives. Highly specific methods have been developed for the detection and assay of the various kinds of protein modifications. Because the generation of carbonyl derivatives occurs by many different mechanisms, the level of carbonyl groups in proteins is widely used as a marker of oxidative protein damage. The level of oxidized proteins increases with aging and in a number of age-related diseases. However, the accumulation of oxidized protein is a complex function of the rates of ROS formation, antioxidant levels, and the ability to proteolytically eliminate oxidized forms of proteins. Thus, the accumulation of oxidized proteins is also dependent upon genetic factors and individual life styles. It is noteworthy that surface-exposed methionine and cysteine residues of proteins are particularly sensitive to oxidation by almost all forms of ROS; however, unlike other kinds of oxidation the oxidation of these sulfur-containing amino acid residues is reversible. It is thus evident that the cyclic oxidation and reduction of the sulfur-containing amino acids may serve as an important antioxidant mechanism, and also that these reversible oxidations may provide an important mechanism for the regulation of some enzyme functions.
Keywords: Keywords: Protein carbonyls – Oxygen free radicals – Methionine oxidation/reduction – Oxidized protein proteolysis

Recent advances in the analysis of oxidized proteins by J. R. Requena; R. L. Levine; E. R. Stadtman (221-226).
 Glutamic semialdehyde is a product of oxidation of arginine and proline, and aminoadipic semialdehyde, of oxidation of lysine. These two carbonyl-containing compounds are the main carbonyl products of metal-catalyzed oxidation of proteins, accounting for 55–100% of the total carbonyl value. Accordingly, they are quantitatively very important contributors to the total value of protein carbonyls in tissues as measured by the classic spectophotometric assay. Sensitive gas chromatography-mass spectrometry based analytical methods allow their quantitation in a variety of biological samples, including tissue protein, cell cultures and lipoproteins. These measurements provide specific information on the oxidative status of proteins that is complementary to that afforded by protein carbonyls, and will be useful tools in the ongoing effort to define and assess the role of protein oxidation in pathology and aging.
Keywords: Keywords: Glutamic semialdehyde – Aminoadipic semialdehyde – Hydroxyaminovaleric acid – Hydroxyaminocaproic acid – Protein carbonyls – Metal catalyzed oxidation

Tyrosine oxidation products: analysis and biological relevance by C. Giulivi; N. J. Traaseth; K. J. A. Davies (227-232).
 Dityrosine is found in several proteins as a product of UV irradiation, γ-irradiation, aging, exposure to oxygen free radicals, nitrogen dioxide, peroxynitrite, and lipid hydroperoxides. Interest of dityrosine in proteins is based on its potential as a specific marker for oxidatively damaged proteins and their selective proteolysis, hence it could be used as a marker for oxidative stress. Dityrosine is also the product of normal post-translational processes affecting specific structural proteins. Since post-translational modification of a given amino acid in a protein is equivalent to the substitution of that residue by an analogue, it has been proposed that the covalent modification of amino acids may serve as a “marking” step for protein degradation.
Keywords: Keywords: Dityrosine – Oxygen free radicals – Proteolysis – Oxidative stress – Protein damage

Dityrosine as a product of oxidative stress and fluorescent probe by D. A. Malencik; S. R. Anderson (233-247).
 Dityrosine can be a natural component of protein structure, a product of environmental stress, or a product of in vitro protein modification. It is both a cross-link and a fluorescent probe that reports structural and functional information on the cross-linked protein molecule. Diverse reactions produce tyrosyl radicals, which in turn may couple to yield dityrosine. Identification and quantitation of dityrosine in protein hydrolysates usually employs reversed phase high pressure liquid chromatography (RP-HPLC) or gas chromatography. RP-HPLC of protein hydrolysates that have been derivatized with dabsyl chloride gives a complete amino acid analysis that includes dityrosine and 3-nitrotyrosine. Calmodulin, which contains a single pair of tyrosyl residues, undergoes both photoactivated and enzyme-catalyzed dityrosine formation. Polarization measurements, employing the intrinsic fluorescence of dityrosine, and catalytic activity determinations show how different patterns of inter- and intramolecular cross-linking affect the interactions of calmodulin with Ca2+ and enzymes.
Keywords: Keywords: Dityrosine – Tyrosyl radicals – Protein oxidation – Cross-linking – Calmodulin – 3-Nitrotyrosine

 Histidine and lysine are two representative targets of oxidative modifications. Histidine is extremely sensitive to a metal-catalyzed oxidation, generating 2-oxo-histidine and its ring-ruptured products, whereas the oxidation of lysine generates carbonyl products, such as aminoadipic semialdehyde. On the other hand, both histidine and lysine are nucleophilic amino acids and therefore vulnerable to modification by lipid peroxidation-derived electrophiles, such as 2-alkenals, 4-hydroxy-2-alkenals, and ketoaldehydes, derived from lipid peroxidation. Histidine shows specific reactivity toward 2-alkenals and 4-hydroxy-2-alkenals, whereas lysine is a ubiquitous target of aldehydes, generating various types of adducts. Covalent binding of reactive aldehydes to histidine and lysine is associated with the appearance of carbonyl reactivity and antigenecity of proteins.
Keywords: Keywords: Oxidative modification of protein – Lipid peroxidation – Reactive aldehydes

Hypochlorite-induced oxidation of amino acids, peptides and proteins by C. L. Hawkins; D. I. Pattison; M. J. Davies (259-274).
 Activated phagocytes generate the potent oxidant hypochlorite (HOCl) via the release of the enzyme myeloperoxidase and hydrogen peroxide. HOCl is known to react with a number of biological targets including proteins, DNA, lipids and cholesterol. Proteins are likely to be major targets for reaction with HOCl within a cell due to their abundance and high reactivity with HOCl. This review summarizes information on the rate of reaction of HOCl with proteins, the nature of the intermediates formed, the mechanisms involved in protein oxidation and the products of these reactions. The predicted targets for reaction with HOCl from kinetic modeling studies and the consequences of HOCl-induced protein oxidation are also discussed.
Keywords: Keywords: Hypochlorite – Myeloperoxidase – Protein oxidation – Chloramines – Chloramides – Radicals – Oxidative stress – Fragmentation

 The chemical modification of protein by nonenzymatic browning or Maillard reactions increases with age and in disease. Maillard products are formed by reactions of both carbohydrate- and lipid-derived intermediates with proteins, leading to formation of advanced glycation and lipoxidation end-products (AGE/ALEs). These modifications and other oxidative modifications of amino acids increase together in proteins and are indicators of tissue aging and pathology. In this review, we describe the major pathways and characteristic products of chemical modification of proteins by carbohydrates and lipids during the Maillard reactions and identify major intersections between these pathways. We also describe a new class of intracellular sulfhydryl modifications, Cys-AGE/ALEs, that may play an important role in regulatory biology and represent a primitive link between nonenzymatic and enzymatic chemistry in biological systems.
Keywords: Keywords: Advanced glycation end-product (AGE) – Advanced lipoxidation end-product (ALE) – Nε-(carboxymethyl)lysine – Glyoxal – Maillard reaction – Methylglyoxal

Scavenger receptors for oxidized and glycated proteins by S. Horiuchi; Y. Sakamoto; M. Sakai (283-292).
 Our present knowledge on chemically modified proteins and their receptor systems is originated from a proposal by Goldstein and Brown in 1979 for the receptor for acetylated LDL which is involved in foam cell formation, one of critical steps in atherogenesis. Subsequent extensive studies using oxidized LDL (OxLDL) as a representative ligand disclosed at least 11 different scavenger receptors which are collectively categorized as “scavenger receptor family”. Advanced glycation endproducts (AGE) and their receptor systems have been studied independently until recent findings that AGE-proteins are also recognized as active ligands by scavenger receptors including class A scavenger receptor (SR-A), class B scavenger receptors such as CD36 and SR-BI, type D scavenger receptor (LOX-1) and FEEL-1/FEEL-2. Three messages can be summarized from these experiments; (i) endocytic uptake of OxLDL and AGE-proteins by macrophages or macrophage-derived cells is mainly mediated by SR-A and CD36, which is an important step for foam cell formation in the early stage of atherosclerosis, (ii) selective uptake of cholesteryl esters of high density lipoprotein (HDL) mediated by SR-BI is inhibited by AGE-proteins, suggesting a potential pathological role of AGE in a HDL-mediated reverse cholesterol transport system, (iii) a novel scavenger receptor is involved in hepatic clearance of plasma OxLDL and AGE-proteins.
Keywords: Keywords: Scarenger receptors – Modified LDL – Advanced glycation endproducts (AGE) – Macrophage – Foam cell – Cholesterol transport – Atherosclerosis

Peroxynitrite reactivity with amino acids and proteins by B. Alvarez; R. Radi (295-311).
Peroxynitrite, the product of the fast reaction between nitric oxide (NO) and superoxide O2 •− radicals, is an oxidizing and nitrating agent which is able to traverse biological membranes. The reaction of peroxynitrite with proteins occurs through three possible pathways. First, peroxynitrite reacts directly with cysteine, methionine and tryptophan residues. Second, peroxynitrite reacts fast with transition metal centers and selenium-containing amino acids. Third, secondary free radicals arising from peroxynitrite homolysis such as hydroxyl and nitrogen dioxide, and the carbonate radical formed in the presence of carbon dioxide, react with protein moieties too. Nitration of tyrosine residues is being recognized as a marker of the contribution of nitric oxide to oxidative damage. Peroxynitrite-dependent tyrosine nitration is likely to occur through the initial reaction of peroxynitrite with carbon dioxide or metal centers leading to secondary nitrating species. The preferential protein targets of peroxynitrite and the role of proteins in peroxynitrite detoxifying pathways are discussed.
Keywords: Keywords: Peroxynitrite – Amino acids – Cysteine – Nitrotyrosine – Nitric oxide – Superoxide

Thiolation and nitrosation of cysteines in biological fluids and cells by P. Di Simplicio; F. Franconi; S. Frosalí; D. Di Giuseppe (323-339).
 Thiols (RSH) are potent nucleophilic agents, the rates of which depend on the pKa of the sulfhydryl. Unlike compounds having other nucleophile moieties (–OH or –NH2), RSH are involved in reactions, such as conjugations, redox and exchange reactions. Although protein SH groups (PSH) react like non-protein thiols (NPSH), the biochemistry of proteins is much more complex for reasons such as steric hindrance, charge distribution and accessibility of PSH to the solvent (protein conformation). The reaction rates and types of end-products of PSH vary a lot from protein to protein. The biological problem is even more complex because in all compartments and tissues, there may be specific competition between thiols (namely between GSH and PSH), regulated by the properties of antioxidant enzymes. Moreover, PSH are divided biologically into essential and non-essential and their respective influence in the various biological systems is unknown. It follows that during phenomena eliciting a prompt thiol response (oxidative stress), the antioxidant PSH response and reaction mechanisms vary considerably from case to case. For example, in spite of a relatively low pKa that should guarantee good antioxidant capacity, PSH of albumin has much less propensity to form adducts with conjugating agents than NPSH; moreover, the structural characteristics of the protein prevent albumin from forming protein disulfides when exposed to oxidants (whereas protein-thiol mixed disulfides are formed in relative abundance). On the other hand, proteins with a relatively high reactivity, such rat hemoglobin, have much greater antioxidant capacity than GSH, but although human hemoglobin has a pKa similar to GSH, for structural reasons it has less antioxidant capacity than GSH.When essential PSH are involved in S-thiolation and S-nitrosation reactions, a similar change in biological activity is observed. S-thiolated proteins are a recurrent phenomenon in oxidative stress elicited by reactive oxygen species (ROS). This event may be mediated by disulfides, that exchange with PSH, or by the protein intermediate sulfenic acid that reacts with thiols to form protein-mixed disulfides. During nitrosative stress elicited by reactive nitrogen species (RNS), depending on the oxygen concentration of the system, nitrosation reactions of thiols may also be accompanied by protein S-thiolation. In this review we discuss a number of cell processes and biochemical modifications of enzymes that indicate that S-thiolation and S-nitrosation may occur simultaneously in the same protein in the presence of appropriate interactions between ROS and RNS.
Keywords: Keywords: Thiols – Nitrosothiols – Protein SH groups – NO – S-Thiolation – S-Nitrosylation

Our understanding of in vivo tyrosine nitration has been confounded by problems associated with the analytical approaches that have been employed to quantify 3-nitroyrosine (3-NT). Trace analysis is a demanding task under the best of circumstances, but 3-NT offers some special concerns. This review examines some of these concerns and discusses approaches to ensuring that carefully validated analytical data are generated.
Keywords: Keywords: 3-Nitrotyrosine – Trace analysis – Artifactual formation – Assay validation – Mass spectrometry

Amino acid and protein oxidation in cardiovascular disease by M.-L. Brennan; S. L. Hazen (365-374).
 Substantial evidence suggests that oxidative events contribute to the pathogenesis of atherosclerotic heart disease. For example, animal model data and numerous in vitro studies point to specific pathways as participants in disease initiation and progression. Moreover, recent clinical studies demonstrate clinical utility in monitoring systemic levels of protein-bound nitrotyrosine as a predictor of risk for coronary artery disease, atherosclerotic burden, and response to statin therapy. However, a definitive cause-and-effect relationship between oxidation and atherosclerosis has yet to be established, and multiple recent large prospective “antioxidant” intervention trials have failed to significantly impact upon disease risk and progression. In this review we highlight why such failures should not be taken as an indictment of the “Oxidation Hypothesis.” Emphasis will be placed on discussion of molecular markers whose structures convey information about oxidation pathways leading to their formation, and which appear to be mechanistically linked to the disease process. Only through rational design of targeted interventions aimed at suppressing distinct oxidation pathways, with concomitant monitoring of antioxidant efficacy in human clinical studies, will answers to the role of oxidation in the pathogenesis of human atherosclerosis be defined.
Keywords: Keywords: Atherosclerosis – Inflammation – Lipid peroxidation – Myeloperoxidase – Nitration – Chlorination – Free Radical

Protein oxidation at the air-lung interface by F. J. Kelly; I. S. Mudway (375-396).
 Whilst performing its normal functions the lung is required to deal with a range of toxic insults. Whether these are infectious agents, allergens or air pollutants they subject the lung to a range of direct and indirect oxidative stresses. In many instances these challenges lead to oxidative alterations of peptides and proteins within the lung. Measurement of protein oxidation products permits the degree of oxidative stress to be assessed and indicates that endogenous antioxidant defences are overwhelmed. The range of protein oxidation products observed is diverse and the nature and extent of specific oxidation products may inform us about the nature of the damaging ROS and NOS. Recently, there has been a significant shift away from the measurement of these oxidation products simply to establish the presence of oxidative stress, to a focus on identifying specific proteins sensitive to oxidation and establishing the functional consequences of these modifications. In addition the identification of specific enzyme systems to repair these oxidative modifications has lead to the belief that protein function may be regulated through these oxidation reactions. In this review we focus primarily on the soluble protein components of within the surface liquid layer in the lung and the consequence of their undue oxidation.
Keywords: Keywords: Respiratory tract lining fluid – Lung – Protein carbonyl – Air pollution – α-tocopherol – Ascorbic acid – Glutathione

Protein oxidation in aging: endoplasmic reticulum as a target by D. van der Vlies; J. Woudenberg; J. A. Post (397-407).

Homocysteine and oxidative stress by A. F. Perna; D. Ingrosso; N. G. De Santo (409-417).
 Hyperhomocysteinemia is an independent risk factor for cardiovascular disease (ischemic disease, such as stroke and myocardial infarction, and arterial and venous thrombotic events) in the general population. We can assume that the association is causal, based on the example of homocystinuria, and on the evidence put forward by several basic science and epidemiological studies; however, the results of large intervention trials, which will grant further support to this hypothesis, are not yet available. In addition, the mechanisms underlying this relationship, and also explaining the several toxic effects of homocysteine, related or not to cardiovascular disease, are unclear. Oxidation is one of the most favored postulated mechanisms; others are nitrosylation, acylation, and hypomethylation. Regarding the relative importance of these mechanisms, each of these hold pros and cons, and these are weighed in order to propose a balance of evidence.
Keywords: Keywords: Homocysteine – Homocystinuria – Cardiovascular risk – Mechanisms of toxicity – Uremia – Chronic renal failure

 Proteomics offers the opportunity elucidate the complex protein interactions of cellular systems by studying the products of genes, i.e., proteins, and their structure, function and localization. The purpose of proteomics is to explain the information contained in the genome sequences in order to provide clues on cellular events, especially related to disease.Our proteomic approach has made possible the identification of specifically oxidized proteins in Alzheimer’s disease (AD) brain, providing for the first time evidence on how oxidative stress plays a crucial role in AD-related neurodegeneration. This represents an example of the use of proteomics to solve biological problems related to disease. The field, which is still in its infancy, represents a very promising way to elucidate mechanism of disease at a protein level. However, the techniques that support its development present several limitations and require introduction of new tools and innovation in order to achieve a fast, reliable and sensitive method to understand normal biological processes and their regulation as well as these cellular properties in disease.
Keywords: Keywords: Proteomics – Alzheimer’s disease – Oxidized proteins – Neurodegeneration

 This review has focused on the evidence for the involvement of nitrative oxidation in certain neurodegenerative disorders (Parkinson’s Disease, Alzheimer’s Disease, Amyotrophic Lateral Sclerosis), stroke, and inflammatory and autoimmune disorders (with particular attention devoted to multiple sclerosis).The relationship between protein peroxidation and pathological changes observed in the above disorders has been reported. Whereas many of the findings are from studies with animal models and autoptic specimens from human patients, few data are available from cerebrospinal fluid and blood samples of the patients at different times and disease stages.The participation of nitrative oxidation to the direct and indirect injury of neurons and other cells of the brain (i.e., oligodendrocytes, for multiple sclerosis) is clear; less evident is their relevance for the development and progression of these disorders.Further studies should be aimed to establish the clinical and prognostic value of peroxidative markers for the CNS diseases considered. This is fundamental for the development of therapeutic interventions antagonizing nitric oxide-related species damage.
Keywords: Keywords: Protein nitration – Nitrotyrosine – Neurodegenerative diseases – Inflammatory and autoimmune diseases of the central nervous system – Stroke – Pathogenic mechanisms

Redox regulation of heat shock protein expression in aging and neurodegenerative disorders associated with oxidative stress: A nutritional approach by V. Calabrese; G. Scapagnini; C. Colombrita; A. Ravagna; G. Pennisi; A. M. Giuffrida Stella; F. Galli; D. A. Butterfield (437-444).
Oxidative stress has been implicated in mechanisms leading to neuronal cell injury in various pathological states of the brain. Alzheimer’s disease (AD) is a progressive disorder with cognitive and memory decline, speech loss, personality changes and synapse loss. Many approaches have been undertaken to understand AD, but the heterogeneity of the etiologic factors makes it difficult to define the clinically most important factor determining the onset and progression of the disease. However, increasing evidence indicates that factors such as oxidative stress and disturbed protein metabolism and their interaction in a vicious cycle are central to AD pathogenesis.Brains of AD patients undergo many changes, such as disruption of protein synthesis and degradation, classically associated with the heat shock response, which is one form of stress response. Heat shock proteins are proteins serving as molecular chaperones involved in the protection of cells from various forms of stress.Recently, the involvement of the heme oxygenase (HO) pathway in anti-degenerative mechanisms operating in AD has received considerable attention, as it has been demonstrated that the expression of HO is closely related to that of amyloid precursor protein (APP). HO induction occurs together with the induction of other HSPs during various physiopathological conditions. The vasoactive molecule carbon monoxide and the potent antioxidant bilirubin, products of HO-catalyzed reaction, represent a protective system potentially active against brain oxidative injury. Given the broad cytoprotective properties of the heat shock response there is now strong interest in discovering and developing pharmacological agents capable of inducing the heat shock response.Increasing interest has been focused on identifying dietary compounds that can inhibit, retard or reverse the multi-stage pathophysiological events underlying AD pathology. Alzheimer’s disease, in fact, involves a chronic inflammatory response associated with both brain injury and β-amyloid associated pathology. All of the above evidence suggests that stimulation of various repair pathways by mild stress has significant effects on delaying the onset of various age-associated alterations in cells, tissues and organisms. Spice and herbs contain phenolic substances with potent antioxidative and chemopreventive properties, and it is generally assumed that the phenol moiety is responsible for the antioxidant activity. In particular, curcumin, a powerful antioxidant derived from the curry spice turmeric, has emerged as a strong inducer of the heat shock response. In light of this finding, curcumin supplementation has been recently considered as an alternative, nutritional approach to reduce oxidative damage and amyloid pathology associated with AD. Here we review the importance of the heme oxygenase pathway in brain stress tolerance and its significance as an antidegenerative mechanism potentially important in AD pathogenesis. These findings have offered new perspectives in medicine and pharmacology, as molecules inducing this defense mechanism appear to be possible candidates for novel cytoprotective strategies. In particular, manipulation of endogenous cellular defense mechanisms such as the heat shock response, through nutritional antioxidants or pharmacological compounds, represents an innovative approach to therapeutic intervention in diseases causing tissue damage, such as neurodegeneration. Consistent with this notion, maintenance or recovery of the activity of vitagenes, such as the HO gene, conceivably may delay the aging process and decrease the occurrence of age-related neurodegenerative diseases.
Keywords: Keywords: Oxidative stress – AD pathogenesis – Heme oxygenase pathway – Vitamin E – Vitamin C – Micronutrients – Antioxidants