Protein S-thiolation Research Articles

Protein S-Thiolation in Hepatocytes Stimulated by t-Butyl Hydroperoxide, Menadione, and Neutrophils

In order to examine potentially important S-thiolated proteins, 35S-labeled hepatocytes were exposed to oxidative stress. A similar group of S-thiolated proteins including carbonic anhydrase III was observed in cells treated with t-butyl hydroperoxide, menadione, or stimulated neutrophils. The radioactive thiols bound to hepatocyte proteins were identified by HPLC and more than 85% was glutathione. In menadione-treated hepatocytes, proteins were gradually S-thiolated over 30 min and 25% of the cellular glutathione pool became protein-bound. In t-butyl hydroperoxide-treated cells, S-thiolation was more transient and 11% of the glutathione was protein-bound. Neutrophil-treated hepatocytes had nearly the same amount of protein S-thiolation (8% after 25 min). Two major proteins that were S-thiolated in untreated hepatocytes did not increase during any form of oxidative stress. In neutrophil-treated hepatocytes protein S-thiolation was not accompanied by either formation of glutathione disulfide or a measureable change in the total amount of glutathione. In both t-butyl hydroperoxide- and menadione-treated cells there was extensive formation of glutathione disulfide and in menadione-treated cells a significant increase in the total hepatocyte glutathione pool was observed. This result suggests that protein S-thiolation may occur by mechanisms that do not result from thiol/disulfide exchange between glutathione disulfide and protein sulfhydryls. It is suggested that a thiyl radical intermediate is important in neutrophil-mediated protein S-thiolation.

Oxidative stress induces S-thiolation of specific proteins in cultured gastric mucosal cells.

Oxidative stress induces the formation of protein-mixed disulfides with low-molecular-weight thiols, especially glutathione. We analyzed this process, termed S-thiolation, in cultured gastric mucosal cells from guinea pigs by gel electrophoresis and autoradiography after radiolabeling of the intracellular glutathione pool with 35S. Hydrogen peroxide (H2O2) or diamide initiated rapid and reversible S-thiolation of specific proteins with molecular masses of 42, 30, 29, 28, and 22 kDa. Diamide caused particularly prominent S-thiolation of the 42 kDa protein. This protein was identified as actin by immunoblot analysis and actin-myosin precipitation. Fluorescence microscopy revealed that diamide caused a disappearance of normal stress fibers and a concomitant increase in actin polymerization in association with contraction of the cells. These morphological changes were completely reversible within minutes. With cells depleted of glutathione by incubation with DL-buthionine-[S,R]-sulfoximine, diamide caused severe contraction and rounding, and the cells detached from the culture plates. S-thiolation of actin could help protect gastric mucosal cells against irreversible organization of microfilaments by preserving microfilament dynamics under oxidative stress.

42] Protein S-thiolation and dethiolation

Publisher Summary S-Thiolated proteins—mixed disulfides of proteins and low molecular weight thiols—are very early products of protein oxidation during oxidative stress, occurring within seconds after the generation of oxygen radicals. As a result, assessment of the extent and specificity of this process during oxidative stress is one of the best measures of the primary effects of oxygen radical generation on intact cells. The development of methods for measuring the S-thiolation status of individual proteins, eventually studying organs of intact animals and even humans, is essential for a more complete understanding of the role of oxidative stress in human disease. This chapter introduces a method to quantify the S-thiolation status of mixed cellular proteins in intact cells and second method to determine the activity of the cellular reductive processes—namely, dethiolases. This method tests the effects of toxic materials on protein S-thiolation. Protein dethiolation can be measured with S-thiolated substrate proteins containing a radioactive low molecular weight thiol.

Protein-specific S-thiolation in human endothelial cells during oxidative stress

Confluent human umbilical vein endothelial cells were treated with diamide, t-butyl hydroperoxide t-BH) or the hydrogen peroxide generating system glucose/glucose oxidase and the effects on glutathione oxidation and protein S-thiolation were examined. In the presence of all three oxidants glutathione was rapidly oxidized to a similar extent and S-thiolation of a limited number of proteins occurred. Diamide caused considerable S-thiolation of proteins with molecular masses of 44, 34, 24 and 14 kDa, of which the protein with molecular mass of 44 kDa was most extensively modified. f-BH caused extensive modification of proteins with molecular masses of 24 and 14 kDa whilst hydrogen peroxide caused S-thiolation of proteins of 39, 24 and 14 kDa. This study shows that S-thiolation of proteins is an important metabolic response to oxidant insult in human endothelial cells and that the specificity of the response depends on the chemical nature of the oxidant.

Phagocytosis and stimulation of the respiratory burst by phorbol diester initiate S-thiolation of specific proteins in macrophages.

Addition of chemical oxidants to cells in culture has been shown to induce binding of low-molecular-weight thiols to reactive sulfhydryls on proteins in a process termed S-thiolation. We found that stimulation of the respiratory burst in mouse macrophages, with release of O2-, resulted in S-thiolation of several proteins, most prominently three with molecular weights of 74, 33, and 22 kDa. One protein (28 kDa) was S-thiolated without addition of an exogenous stimulus. Exposure of cells to concentrations of hydrogen peroxide like those released in the respiratory burst induced S-thiolation of these same proteins. S-thiolation and release of O2- began at approximately the same time. Stimulation of LPS-elicited macrophages induced prominent S-thiolation of three different proteins (38, 30, and 21 kDa). Under the conditions of these experiments, there was no detectable increase in glutathione disulfide and a negligible decrease in glutathione, which suggests that S-thiolation can occur without significant perturbation of the glutathione peroxidase/reductase cycle. S-thiolation of proteins could help protect the macrophage against the autoxidative damage associated with the respiratory burst. Modification of specific proteins by S-thiolation might serve to modulate cellular metabolic events.

Phosphorylase and creatine kinase modification by thiol-disulfide exchange and by xanthine oxidase-initiated S-thiolation

The reaction of glycogen phosphorylase b and creatine kinase with glutathione disulfide, cystine, and cystamine was compared by direct analysis on electrofocusing gels. This method was useful for individual proteins or for mixtures of the proteins. Millimolar concentrations of glutathione disulfide were required for both proteins and the rate of modification of each protein was similar. The reaction of glutathione disulfide with creatine kinase was inhibited by reduced glutathione (GSH), but the effect on the reaction with phosphorylase was minimal. Cystine and cystamine were required in micromolar amounts to effectively form the disulfide adducts. Both proteins were modified by cystine but cystamine reacted only with phosphorylase. Cystamine (10 μ m) was an effective inhibitor of the reaction of phosphorylase b with 2 m m glutathione disulfide. S-thiolation of creatine kinase inactivated the enzyme and a direct assay of the enzyme activity could be used to quantitate S-thiolation of this protein by each of the disulfides. The effect of each disulfide on enzyme activity confirmed the results obtained by gel electrofocusing. Glutathione disulfide and cystine both inactivated the enzyme while cystamine had no effect on the activity. S-thiolation of phosphorylase had no observable effect on any activity parameter, but it effectively prevented binding of phosphorylase to high-molecular-weight glycogen, probably at the glycogen storage site of phosphorylase. The rate of S-thiolation of a mixture of phosphorylase and creatine kinase by thiol-disulfide exchange with glutathione disulfide was compared to the rate of S-thiolation of these proteins by a xanthine oxidase-initiated process (presumably due to protein sulfhydryl activation by reactive oxygen species). The xanthine oxidase-initiated mechanism was somewhat faster than thiol-disulfide exchange with both proteins. It was shown that GSH inhibited S-thiolation of creatine kinase by this mechanism as well as by thiol-disulfide exchange. It is suggested that both mechanisms may play a role in protein S-thiolation in vivo. For proteins that are typified by creatine kinase, the concentration of GSH in the cells may determine whether the S-thiolated form of the protein accumulates. For proteins typified by phosphorylase b, the accumulation of S-thiolated forms may be more independent of GSH.

Protein S-thiolation in cultured hepatocytes

Protein S-thiolation in cultured hepatocytes

The respiratory burst induces S-thiolation of specific proteins in human neutrophils

The respiratory burst induces S-thiolation of specific proteins in human neutrophils

Role of specific protein S-thiolation under oxidative stress

Role of specific protein S-thiolation under oxidative stress

Specific S-thiolation of a 30-kDa cytosolic protein from rat liver under oxidative stress.

Thin-gel isoelectric focusing (IEF) is a simple and sensitive method of quantifying S-thiolation of individual proteins (protein mixed-disulfide formation). IEF of rat liver cytosol identified one major protein (pI 7.0) which underwent S-thiolation with glutathione disulfide to produce two acidic bands with pIs 6.4 and 6.1. The S-thiolated forms of the protein were purified by preparative isoelectric focusing. An apparent molecular mass of 30 kDa was determined by SDS/polyacrylamide gel electrophoresis. The 30-kDa protein amounted to 7 +/- 2% of the total cytosolic protein on IEF. The most abundant soluble protein of freshly isolated hepatocytes, with an identical isoelectric point to the liver 30-kDa protein, was modified in a similar manner in response to oxidative stress induced by model compounds. Addition of 50 microM tert-butyl hydroperoxide, 50 microM diamide [1,1-azobis(N,N'-dimethylformamide)] or 20 microM menadione (2-methyl-1,4-naphthoquinone) initiated the S-thiolation within less than 2 min in the hepatocytes. These compounds, at the concentrations employed, did not result in cell death. Menadione produced slowly progressive S-thiolation of the protein, while tert-butyl hydroperoxide or diamide produced rapid S-thiolation that decreased quickly after 2 min.

S-thiolation of creatine kinase and glycogen phosphorylase b initiated by partially reduced oxygen species

S-thiolation of cardiac creatine kinase and skeletal muscle glycogen phosphorylase b was initiated by reduced oxygen species in reaction mixtures containing reduced glutathione. Both proteins were extensively modified at similar rates under conditions in which the oxidation of glutathione was inadequate to cause S-thiolation by thiol-disulfide exchange. Creatine kinase was both S-thiolated and non-reducibly oxidized at the same time at low glutathione concentration. The amount of each modification was decreased by adding additional reduced glutathione, and with adequate glutathione oxidation was prevented while S-thiolation was still very active. S-thiolation of glycogen phosphorylase b was not significantly affected by glutathione concentration and non-reducible oxidation of glycogen phosphorylase b was not observed. These experiments suggest that oxyradical or H 2O 2-initiated processes may be an important mechanism of protein S-thiolation during oxidative stress, and that the cellular concentration of glutathione may be an important factor in S-thiolation of different proteins. Both creatine kinase and glycogen phosphorylase b competed favorably with ferricytochrome c for superoxide anion in the standard xanthine oxidase system for the generation of oxyradicals and H 2O 2. These proteins were as effective as ascorbate and much more effective than reduced glutathione in this regard. Ascorbate was also an effective inhibitor of oxyradical-initiated S-thiolation of creatine kinase, suggesting a role of superoxide anion in protein S-thiolation. Other experiments showed that both catalase and superoxide dismutase could partially inhibit protein S-thiolation. Thus, reduced oxygen species may react with protein sulfhydryls resulting in S-thiolation by a mechanism that involves the reaction of an activated protein thiol with reduced glutathione.

Oxy radical-initiated protein S-thiolation and enzymic dethiolation.

It has often been suggested that protein S-thiolation/dethiolation (Figure 1) is an important form of metabolic regulation 1,2 . This suggestion usually arises from experiments in which a thiol-disulfide exchange mechanism is considered to be the basis for this process. Since cellular glutathione is normally very reduced, the thiol-disulfide exchange mechanism requires a significant oxidation of this thiol to account for appreciable protein modification in vivo. In addition, both S-thiolation and dethiolation by thiol-disulfide exchange probably require catalysis to occur at significant rates in vivo. These considerations make thiol-disulfide exchange less appealing than a direct oxy radical-initiated mechanism of S-thiolation. This paper describes experiments with electrofocusing technology to assess various mechanisms of protein S-thiolation and dethiolation.

S-thiolation of cytoplasmic cardiac creatine kinase in heart cells treated with diamide

Two methods for quantitation of protein S-thiolation, by isoelectric focusing or by enzyme activity, were used for studying S-thiolation of cytoplasmic cardiac creatine kinase. With these methods, creatine kinase was identified as a major S-thiolated protein in both bovine and rat heart. In rat heart cell cultures, creatine kinase became 10% S-thiolated during a 10 min incubation with 0.2 mM diamide. This enzyme became S-thiolated more slowly than other heart cell proteins and it also dethiolated more slowly. Two sequential additions of diamide at a 25 min interval caused twice as much S-thiolation after the second addition as compared to the first. This increased sensitivity to the second diamide treatment may have resulted from glutathione loss during the first addition which produced a higher GSSG-to-GSH ratio after the second treatment. The GSSG-to-GSH ratio was highest prior to the maximum S-thiolation of creatine kinase, but, in general, the time course of glutathione was similar to the S-thiolation of creatine kinase. This study demonstrates that cytoplasmic creatine kinase is S-thiolated and, therefore, inhibited during a diamide-induced oxidative stress in heart cells. Implications for regulation of cardiac metabolism during oxidative stress are discussed.

Hormones, glutathione status and protein S-thiolation

The formation of mixed disulfides between proteins and glutathione has been discussed as a potentially interesting metabolic signal. The S-thiolation of proteins with glutathione has been observed in several systems in vitro. We have correlated the increase in glutathione disulfide (GSSG) with the amount of protein mixed disulfides. The methodological aspects are briefly presented; normal values for protSSG are about 20–30 nmol per g wet weight of liver. Several processes have been related to changes in the thiol redox state. The stimulation of flux through the pentose phosphate pathway during the metabolism of t-butyl hydroperoxide is presented, and the increase in cellular activity of glucose-6-phosphate dehydrogenase is correlated with the increase in the level of protSSG. Hormonal stimulation of GSH efflux from the liver by vasopressin or by alpha-adrenergic agonists such as phenylephrine or epinephrine is presented and discussed in relation to physiological states of peripheral (non hepatic) GSH utilization. Preliminary work relates the release of GSH to the perturbations in thiol redox state in inflammation and in exercise.

A thin-gel isoelectric focusing method for quantitation of proteins S-thiolation

A thin-gel isoelectric focusing method has been developed for analysis of protein S-thiolation (formation of mixed disulfides with low molecular weight thiols). The method is rapid and it can be used with 3 to 5 μg of a pure protein, or 15 to 20 μg of tissue extract protein. It is possible to detect a modification of the protein sulfhydryl by either charged or uncharged thiols, and to determine the quantity of different S-thiolated protein species in a modified sample. The method was used to quantitate the amount of S-thiolation of phosphorylase b in a reaction with oxidized glutathione that produced four S-thiolated forms of the enzyme. The method was also used to detect S-thiolation of two proteins in a cardiac tissue extract treated with diamide. One of the protein bands was shown to be S-thiolated with both cysteine and glutathione, while the other band was S-thiolated only with glutathione.

A comparison of protein S-thiolation (protein mixed-disulfide formation) in heart cells treated with t-butyl hydroperoxide or diamide

Beating neonatal heart cell cultures were treated with diamide or t-butyl hydroperoxide, and changes in glutathione oxidation, cell beating, and protein S-thiolation (protein mixed-disulfide formation) were examined. Both compounds caused extensive oxidation of glutathione. Cells treated with diamide stopped beating within 2 min, and beating returned to normal after 30–45 min. Cells stopped beating 25 min after the addition of t-butyl hydroperoxide, and beating did not resume. t-Butyl hydroperoxide caused S-thiolation of a variety of proteins, but only one protein, of molecular mass 23 kDa, was extensively modified. Diamide caused extensive modification of proteins with molecular masses of 97, 42 and 23 kDa as well as three proteins of about 35 kDa. Though the GSSG content of cell cultures returned to normal by 15 min after diamide treatment. S-thiolation of several proteins persisted. These studies show that S-thiolation of proteins is an important metabolic response in cells exposed to an oxidative challenge by t-butyl hydroperoxide or diamide, and that the specificity of the response depends on the agent used.

Protein mixed-disulfides in cardiac cells. S-thiolation of soluble proteins in response to diamide

Protein mixed-disulfides in cultured rat heart cells were analyzed by gel electrophoresis under conditions that eliminated artifactual formation of these protein derivatives. Protein S-thiolation (protein mixed-disulfide formation) was detectable under normal culture conditions. Diamide oxidized intracelluar glutathione in these cells and produced extensive protein S-thiolation. The specificity of this protein modification indicates a role in the regulation of cardiac metabolism.