Lorenzo Lusini

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.

Preferential transport of glutathione versus glutathione disulfide in rat liver microsomal vesicles.

A bi-directional, saturable transport of glutathione (GSH) was found in rat liver microsomal vesicles. GSH transport could be inhibited by the anion transport blockers flufenamic acid and 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid. A part of GSH taken up by the vesicles was metabolized to glutathione disulfide (GSSG) in the lumen. Microsomal membrane was virtually nonpermeable toward GSSG; accordingly, GSSG generated in the microsomal lumen could hardly exit. Therefore, GSH transport, contrary to previous assumptions, is preferred in the endoplasmic reticulum, and GSSG entrapped and accumulated in the lumen creates the oxidized state of its redox buffer.

Different Metabolizing Ability of Thiol Reactants in Human and Rat Blood BIOCHEMICAL AND PHARMACOLOGICAL IMPLICATIONS

The effect of oxidants, electrophiles, and NO donors in rat or human erythrocytes was analyzed to investigate the influence of protein sulfhydryl groups on the metabolism of these thiol reactants. Oxidant-evoked alterations in thiolic homeostasis were significantly different in the two models; large amounts of glutathione protein mixed disulfides were produced in rat but not in human erythrocytes by treatment with hydroperoxides or diamide. The disappearance of all forms of glutathione (reduced, disulfide, protein mixed disulfide) was induced by menadione only in human erythrocytes. The treatment of rat red blood cells with electrophiles produced glutathione S-conjugates to a much lower extent than in human red blood cells; GSH was only minimally depleted in rat red blood cells. The NO donor S-nitrosocysteine induced a rapid transnitrosation reaction with hemoglobin in rat erythrocytes producing high levels of S-nitrosohemoglobin; this reaction in human red blood cells was negligible. All drugs were cleared more rapidly in rat than in human erythrocytes. Unlike human Hb, rat hemoglobin contains three families of protein SH groups; one of these located at position β125 is directly implicated in the metabolism of thiol reactants. This is thought to influence significantly the biochemical, pharmacological, and toxicological effects of some drugs.

Fast-reacting Thiols in Rat Hemoglobins Can Intercept Damaging Species in Erythrocytes More Efficiently Than Glutathione

The S-conjugation rates of the free-reacting thiols present on each component of rat hemoglobin with 5,5-dithio-bis(2,2-nitrobenzoic acid) (DTNB) have been studied under a variety of conditions. On the basis of their reactivity with DTNB (0.5 mm), three classes of thiols have been defined as follows: fast reacting (fHbSH), with t½ <100 ms; slow reacting (sHbSH), with t½ 30–50 s; and very slow reacting (vsHbSH), with t½ 180–270 s. Under paraphysiological conditions, fHbSH (identified with Cys-125β(H3)) conjugates with DTNB 100 times faster than glutathione and ∼4000 times more rapidly than (v)sHbSH (Cys-13α(A11) and Cys-93β(F9)). Such characteristics of fHbSH reactivity that are independent of the quaternary state of hemoglobin are mainly due to the following: (i) its low pK (∼6.9, the cysteinyl anion being stabilized by a hydrogen bond with Ser-123β(H1)) and (ii) the large exposure to the solvent (as measured by analysis of a model of the molecular surface) and make these thiols the kinetically preferred groups in rat erythrocytes for S-conjugation. In addition, because of the high cellular concentration (8 mm, i.e. four times that of glutathione), fHbSHs are expected to intercept damaging species in erythrocytes more efficiently than glutathione, thus adding a new physiopathological role (direct involvement in cellular strategies of antioxidant defense) to cysteinyl residues in proteins.

Responses of thiols to an oxidant challenge: differences between blood and tissues in the rat

Treatment of rats with diamide (100 mg/kg i.p.) altered the thiol components of the blood to a very different extent than in tissues (liver, kidney, lung, spleen, heart and testis). A total consumption (10 min) and regeneration (120 min) of blood glutathione (GSH), matched by a parallel increase and decrease in glutathione-protein mixed disulfides (GS-SP) was observed. In contrast, no modification of non-protein SH groups (NPSH) and protein SH groups (PSH), GS-SP and malondialdehyde (MDA) was observed in liver, kidney, lung, testis spleen and heart within same time range. In particular, only glutathione disulfide (GSSG) levels and some activities of antioxidant enzymes were modified to a small extent and in an opposite direction in some organs. For example, GSSG, and glucose-6-phosphate dehydrogenase (G-6-PDH) and catalase (CAT) activities appeared up-regulated in one tissue and down-regulated in another. The least modified organ was the liver, whereas lung and spleen were the most affected (lung, GSSG, significantly increased whereas G-6-PDH, glutaredoxin (GRX), GPX, superoxide dimutase (SOD) levels were significantly lowered; spleen, GSSG and the activity of glutathione reductase (GR), G-6-PDH and glutathione transferase (GST) were significantly decreased). The different responses of erythrocytes and organs to diamide were explained by the high affinity of hemoglobin and by the relatively high potential of thiol regeneration in organs. The rapid reversibility of the process of protein S-thiolation in blood and the small effects in organs leads us to propose the existence of an inter-organ cooperation in the rat that regulates protein S-thiolation in blood. Plasma thiols may well play a role in this process.

The pro-oxidant role of protein SH groups of hemoglobin in rat erythrocytes exposed to menadione.

Menadione is selectively toxic to erythrocytes. Although GSH is considered a primary target of menadione, intraerythrocyte thiolic alterations consequent to menadione exposure are only partially known. In this study alterations of GSH and protein thiols (PSH) and their relationship with methemoglobin formation were investigated in human and rat red blood cells (RBC) exposed to menadione. In both erythrocyte types, menadione caused a marked increase in methemoglobin associated with GSH depletion and increased oxygen consumption. However, in human RBC, GSH formed a conjugate with menadione, whereas, in rat RBC it was converted to GSSG, concomitantly with a loss of protein thiols (corresponding to menadione arylation), and an increase in glutathione-protein mixed disulfides (GS-SP). Such differences were related to the presence of highly reactive cysteines, which characterize rat hemoglobin (cys beta125). In spite of the greater thiol oxidation in rat than in human RBC, methemoglobin formation and the rate of oxygen consumption elicited by menadione in both species were rather similar. Moreover, in repeated experiments under N2 or CO-blocked heme, it was found that menadione conjugation (arylation) in both species was not dependent on the presence of oxygen or the status of heme. Therefore, we assumed that GSH (human RBC) and protein (rat RBC) arylation was equally responsible for increased oxygen consumption and Hb oxidation. Moreover, thiol oxidation of rat RBC was strictly related to methemoglobin formation.