Daniela Giustarini

Protein carbonyl groups as biomarkers of oxidative stress

Oxidative stress, an imbalance toward the pro-oxidant side of the pro-oxidant/antioxidant homeostasis, occurs in several human diseases. Among these diseases are those in which high levels of protein carbonyl (CO) groups have been observed, including Alzheimers disease (AD), rheumatoid arthritis, diabetes, sepsis, chronic renal failure, and respiratory distress syndrome. What relationships might be among high level of protein CO groups, oxidative stress, and diseases remain uncertain.The usage of protein CO groups as biomarkers of oxidative stress has some advantages in comparison with the measurement of other oxidation products because of the relative early formation and the relative stability of carbonylated proteins. Most of the assays for detection of protein CO groups involve derivatisation of the carbonyl group with 2,4-dinitrophenylhydrazine (DNPH), which leads to formation of a stable dinitrophenyl (DNP) hydrazone product. This then can be detected by various means, such as spectrophotometric assay, enzyme-linked immunosorbent assay (ELISA), and one-dimensional or two-dimensional electrophoresis followed by Western blot immunoassay. At present, the measurement of protein CO groups after their derivatisation with DNPH is the most widely utilized measure of protein oxidation.

Protein carbonylation in human diseases

Oxidative modifications of enzymes and structural proteins play a significant role in the aetiology and/or progression of several human diseases. Protein carbonyl content is the most general and well-used biomarker of severe oxidative protein damage. Human diseases associated with protein carbonylation include Alzheimers disease, chronic lung disease, chronic renal failure, diabetes and sepsis. Rapid recent progress in the identification of carbonylated proteins should provide new diagnostic (possibly pre-symptomatic) biomarkers for oxidative damage, and yield basic information to aid the establishment an efficacious antioxidant therapy.

Protein S-glutathionylation: a regulatory device from bacteria to humans

S-Glutathionylation is the specific post-translational modification of protein cysteine residues by the addition of the tripeptide glutathione, the most abundant and important low-molecular-mass thiol within most cell types. Protein S-glutathionylation is promoted by oxidative or nitrosative stress but also occurs in unstressed cells. It can serve to regulate a variety of cellular processes by modulating protein function and to prevent irreversible oxidation of protein thiols. Recent findings support an essential role for S-glutathionylation in the control of cell-signalling pathways associated with viral infections and with tumour necrosis factor-(-induced apoptosis. Glyceraldehyde-3-phosphate dehydrogenase has recently been implicated in the regulation of endothelin-1 synthesis by a novel, S-glutathionylation-based mechanism involving messenger RNA stability. Moreover, recent studies have identified S-glutathionylation as a redox signalling mechanism in plants.

S‐Glutathionylation: from redox regulation of protein functions to human diseases

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) play an integral role in the modulation of several physiological functions but can also be potentially destructive if produced in excessive amounts. Protein cysteinyl thiols appear especially sensitive to ROS/RNS attack. Experimental evidence started to accumulate recently, documenting that S‐glutathionylation occurs in a number of physiologically relevant situations, where it can produce discrete modulatory effects on protein function. The increasing evidence of functional changes resulting from this modification, and the growing number of proteins shown to be S‐glutathionylated both in vitro and in vivo support this contention, and confirm this as an attractive area of research. S‐glutathionylated proteins are now actively investigated with reference to problems of biological interest and as possible biomarkers of human diseases associated with oxidative/nitrosative stress.

Oxidative stress and human diseases: Origin, link, measurement, mechanisms, and biomarkers.

Oxidative stress has been related increasingly to the onset and/or progression of a growing number of human diseases. However, large studies on supplementation with anti-oxidants for prevention or treatment of different pathologies have yielded contradictory and mostly negative results, as documented by numerous meta-analyses and clinical trials. Here we analyze in detail the findings of these studies and discuss major aspects that, in our opinion, are likely to be responsible for these confounding data. With the belief that a clear correlation between disease and oxidative stress is far from being proven for most pathological conditions, our argument focuses on the following points: i) choice of biomarker(s) and/or the biological system(s) for the analyses; ii) pitfalls in pre-analytical and analytical methods for assessing oxidative stress; and iii) scientific misconduct. Eventually, suggestions aiming to obtain more convergent results on this topic are provided.

Reversible S-glutathionylation of Cys374 regulates actin filament formation by inducing structural changes in the actin molecule

S-glutathionylation, the reversible formation of mixed disulphides of cysteinyl residues in target proteins with glutathione, occurs under conditions of oxidative stress; this could be a posttranslational mechanism through which protein function is regulated by the cellular redox status. A novel physiological relevance of actin polymerization regulated by glutathionylation of Cys(374) has been recently suggested. In the present study we showed that glutathionylated actin (GS-actin) has a decreased capacity to polymerize compared to native actin, filament elongation being the polymerization step actually inhibited. Actin polymerizability recovers completely after dethiolation, indicating that S-glutathionylation does not induce any protein denaturation and is therefore a reversible oxidative modification. The increased exposure of hydrophobic regions of protein surface observed upon S-glutathionylation indicates changes in actin conformation. Structural alterations are confirmed by the increased rate of ATP exchange as well as by the decreased susceptibility to proteolysis of the subtilisin cleavage site between Met(47) and Gly(48), in the DNase-I-binding loop of the actin subdomain 2. Structural changes in the surface loop 39-51 induced by S-glutathionylation could influence actin polymerization in view of the involvement of the N-terminal portion of this loop in intermonomer interactions, as predicted by the atomic models of F-actin.

Actin carbonylation: from a simple marker of protein oxidation to relevant signs of severe functional impairment.

The number of protein-bound carbonyl groups is an established marker of protein oxidation. Recent evidence indicates a significant increase in actin carbonyl content in both Alzheimers disease brains and ischemic hearts. The enhancement of actin carbonylation, causing the disruption of the actin cytoskeleton and the loss of the barrier function, has also been found in human colonic cells after exposure to hypochlorous acid (HOCl). Here, the effects of oxidation induced by HOCl on purified actin are presented. Results show that HOCl causes a rapidly increasing yield of carbonyl groups. However, when carbonylation becomes evident, some Cys and Met residues have been already oxidized. Covalent intermolecular cross-linking as well as some noncovalent aggregation of carbonylated actin have been found. The covalent cross-linking, unaffected by reducing and denaturing agents, parallels an increase in dityrosine fluorescence. Moreover, HOCl-mediated oxidation induces the progressive disruption of actin filaments and the inhibition of F-actin formation. The molar ratios of HOCl to actin that lead to inhibition of actin polymerization seem to have effect only on cysteines and methionines. The process that involves oxidation of amino acid side chains with formation of a carbonyl group would occur at an extent of oxidative insult higher than that causing the oxidation of some critical amino acid residues. Therefore, the increase in actin content of carbonyl groups found in vivo would indicate drastic oxidative modification leading to drastic functional impairments.

Nitrite and nitrate measurement by Griess reagent in human plasma: evaluation of interferences and standardization.

Nitrite and nitrate represent the final products of nitric oxide (NO) oxidation pathways, and their hematic concentrations are frequently assessed as an index of systemic NO production. However, their intake with food can influence their levels. Nitrite and nitrate could have a role by producing NO, because nitrite can release NO after reaction with deoxyhemoglobin and dietary nitrate can be reduced substantially to nitrite by commensal bacteria in the oral cavity. Different methods have been applied for nitrite/nitrate detection, with the most commonly used being the spectrophotometric assay based on the Griess reagent. However, a reference methodology for these determinations is still missing and many possible interferences have been reported. This chapter assesses how different experimental conditions can influence the results when detecting nitrite and nitrate in human plasma by the Griess assay and provides a simple method characterized by high reproducibility and minimized interferences by plasma constituents.

Methionine oxidation as a major cause of the functional impairment of oxidized actin

A significant specific increase in the actin carbonyl content has been recently demonstrated in human brain regions severely affected by the Alzheimers disease pathology, in postischemic isolated rat hearts, and in human intestinal cell monolayers following incubation with hypochlorous acid (HOCl). We have very recently shown that exposure of actin to HOCl results in the immediate loss of Cys-374 thiol, oxidation of some methionine residues, and, at higher molar ratios of oxidant to protein, increase in protein carbonyl groups, associated with filament disruption and inhibition of filament formation. In the present work, we have studied the effect of methionine oxidation induced by chloramine-T (CT), which at neutral or slightly alkaline pH oxidizes preferentially Met and Cys residues, on actin filament formation and stability utilizing actin blocked at Cys-374. Methionines at positions 44, 47, and 355, which are the most solvent-exposed methionyl residues in the actin molecule, were found to be the most susceptible to oxidation to the sulfoxide derivative. Met-176, Met-190, Met-227, and Met-269 are the other sites of the oxidative modification. The increase in fluorescence associated with the binding of 8-anilino-1-naphtalene sulfonic acid to hydrophobic regions of the protein reveals that actin surface hydrophobicity increases with oxidation, indicating changes in protein conformation. Structural alterations were confirmed by the decreased susceptibility to proteolysis and by urea denaturation curves. Oxidation of some critical methionines (those at positions 176, 190, and 269) causes a complete inhibition of actin polymerization and severely affects the stability of actin filaments, which rapidly depolymerize. The present results would indicate that the oxidation of some critical methionines disrupts specific noncovalent interactions that normally stabilize the structure of actin filaments. We suggest that the process involving formation of actin carbonyl derivatives would occur at an extent of oxidative insult higher than that causing the oxidation of some critical methionine residues. Therefore, methionine oxidation could be a damaging event preceding the appearance of carbonyl groups on actin and a major cause for the functional impairment of the carbonylated protein recently observed both in vivo and in vitro.

Nitric oxide and S-nitrosothiols in human blood

The hypothesis that endothelial-derived relaxing factor (EDRF) is nitric oxide has stimulated a wealth of research into the significance of this novel intriguing molecule. Given its short life, many storage forms of NO as well as targets have been postulated. Among these, a pool of derivatives of NO (S-nitrosothiols, RSNOs) covalently bound to SH groups of proteins and low molecular weight thiols (e.g., glutathione) have been identified in various biological systems. The importance of RSNOs results from the very similar biological actions exhibited by both NO and RSNOs in vivo as well as in vitro. In particular, it has been observed that in the bloodstream, these molecules are able to provoke vasodilatation with a consequent fall in blood pressure and an antithrombotic effect by inhibition of platelet aggregation. Many hypotheses have been postulated about the biochemical species and the mechanisms involved in these processes, but many aspects have not yet been clarified. In addition, some RSNOs have been recently proposed to be clinical parameters, whose levels may vary under some pathological conditions. The therapeutic utility of RSNOs as an alternative to classic NO donors has also been suggested.Here, we provide a critical analysis of the main reports about the biochemical, physiological, pathological and therapeutic properties of RSNOs in the cardiovascular system. Particular attention is addressed to conflicting results and to discrepancies in the methodologies and models utilized. The numerous unanswered questions concerning the role of RSNOs in the control of vascular tone are discussed.