Cysteine Redox Status Research Articles

Низкомолекулярные аминотиолы в плазме крови при ишемическом инсульте в сочетании с сахарным диабетом 2-го типа

It was found that ischemic stroke (IS) results in decreased levels of a number of reduced forms of low molecular weight aminothiols (LMWTs). The study was aimed to assess the impact of type 2 diabetes mellitus (Т2D) on the total content, reduced forms and redox status of LMWTs in patients with IS. A total of 175 patients with IS in the internal carotid artery basin (the average age was 62 (55–69)) years) were assessed, who were admitted to the Center within the first 10–24 h since the onset of neurological disorder. The index group included 68 patients with IS and T2D (males made up 41.2%). The comparison group consisted of 107 patients with IS and stress hyperglycemia (males made up 57%), and the control group included 31 non-diabetic patients with chronic cerebrovascular disease (CCVD) (males made up 54.8%). The admission plasma levels of LMWTs were assessed by liquid chromatography in all patients. It was found, that IS in patients with T2D was associated with the rapid decrease in total cysteine (tCys), total glutathione (tGSH), total homocysteine (tHcy), reduced glutathione (rGSH), and glutathione redox status (GSH RS), along with the increase in cysteine redox status (Cys RS) and homocysteine redox status (Hcy RS). In contrast to patients with stress hyperglycemia developing during the acute period of IS, patients with T2D had lower tCys, tGSH, and tHcy levels. Thus, GSH RS of 4.06% or lower in the first 24 hours after the IS in patients with T2D was a predictor of poor functional outcome (mRS score was 3 or more 3 weeks after IS).

Redox status of cysteines does not alter functional properties of human dUTPase but the Y54C mutation involved in monogenic diabetes decreases protein stability

Recently it was proposed that the redox status of cysteines acts as a redox switch to regulate both the oligomeric status and the activity of human dUTPase. In a separate report, a human dUTPase point mutation, resulting in a tyrosine to cysteine substitution (Y54C) was identified as the monogenic cause of a rare syndrome associated with diabetes and bone marrow failure. These issues prompt a critical investigation about the potential regulatory role of cysteines in the enzyme. Here we show on the one hand that independently of the redox status of wild-type cysteines, human dUTPase retains its characteristic trimeric assembly and its catalytic activity. On the other hand, the Y54C mutation did not compromise the substrate binding and the catalytic properties of the enzyme at room temperature. The thermal stability of the mutant protein was found to be decreased, which resulted in the loss of 67% of its activity after 90 min incubation at the physiological temperature in contrast to the wild-type enzyme. In addition, the presence or absence of reducing agents had no effect on hDUTY54C activity and stability, although it was confirmed that the introduced cysteine contains a solvent accessible thiol group.

Disturbance of thiol/disulfide aminothiols homeostasis in patients with acute ischemic stroke stroke: Preliminary findings

ObjectivesTo determine the disruption of low-molecular-weight aminothiols (LMWTs: cysteine, cysteinylglycine, homocysteine, and glutathione) homeostasis in blood plasma during the acute and early subacute stages after ischemic stroke. Patients and methodsWe admitted 41 patients with primary large-artery atherosclerosis and cardioembolic stroke in the carotid arteries within the first 6–24 h from the moment of neurologic symptoms development. We included 31 patients with chronic cerebral ischemia in the control group. Total LMWT levels and their reduced forms were measured in blood plasma on the 1st, 3rd, 7th, and 15th days after stroke. ResultsOur study demonstrated a decrease of cysteine and cysteinylglycine reduced forms and an increase of total glutathione and cysteine levels. There were no differences in LMWT levels among stroke subtypes (large-artery atherosclerosis and cardioembolic stroke). The decrease (or increase) in GSH and Hcy redox status on the 3rd day after stroke was associated with severe neurological deficit. Total Hcy (1st day), Cys (3rd day) and CG(7th day) levels were associated with the size of cerebral infarction area. Logistic regression analysis indicated that reduced homocysteine, total cysteinylglycine levels, and cysteine redox status at admission were predictive factors for ischemic stroke occurrence with a probability of 86.2% (p < 0.001). ConclusionsLMWTs may indicate the severity of neurological deficit and the size of the cerebral infarct, and their complex determination can be of diagnostic importance both at an early stage of ischemic stroke development and during its monitoring.

P-45 - Subcellular redox manipulation identifies H2O2 as a key signaling molecule in cell behaviour

Accumulation of reactive oxygen species (ROS) in cells, referred to as oxidative stress, has a direct toxic impact on a number of biomolecules (proteins, lipids and nucleic acids). However, recent findings have shown that ROS can also contribute to bona fide physiological processes, leading to a new paradigm in reversible posttranslational modifications involved in signal transduction, known as redox signaling. We recently found that H2O2 formed highly dynamic gradients during embryonic development1. H2O2 can potentially modify cysteine redox status, and thus protein function. We developed molecular tools to rapidly manipulate H2O2 levels in subcellular compartments, in cell culture and in vivo. That allows us to demonstrate the role of H2O2 in plasma membrane dynamics and specifically in protein palmitoylation and filopodia formation. Physiological consequence for embryonic development will be discussed.

Higher Mediterranean Diet Quality Scores and Lower Body Mass Index Are Associated with a Less-Oxidized Plasma Glutathione and Cysteine Redox Status in Adults

BackgroundBoth systemic redox status and diet quality are associated with risk outcomes in chronic disease. It is not known, however, the extent to which diet quality influences plasma thiol/disulfide redox status. ObjectiveThe purpose of this study was to investigate the influence of diet, as measured by diet quality scores and other dietary factors, on systemic thiol/disulfide redox status. MethodsWe performed a cross-sectional study of 685 working men and women (ages ≥18 y) in Atlanta, GA. Diet was assessed by 3 diet quality scores: the Alternative Healthy Eating Index (AHEI), Dietary Approaches to Stop Hypertension (DASH), and the Mediterranean Diet Score (MDS). We measured concentrations of plasma glutathione (GSH), cysteine, their associated oxidized forms [glutathione disulfide (GSSG) and cystine (CySS), respectively], and their redox potentials (EhGSSG and EhCySS) to determine thiol/disulfide redox status. Linear regression modeling was performed to assess relations between diet and plasma redox after adjustment for age, body mass index (BMI), sex, race, and history of chronic disease. ResultsMDS was positively associated with plasma GSH (β = 0.02; 95% CI: 0.003, 0.03) and total GSH (GSH + GSSG) (β = 0.02; 95% CI: 0.003, 0.03), and inversely associated with the CySS:GSH ratio (β = –0.02; 95% CI: –0.04, –0.004). There were significant independent associations between individual MDS components (dairy, vegetables, fish, and monounsaturated fat intake) and varying plasma redox indexes (P < 0.05). AHEI and DASH diet quality indexes and other diet factors of interest were not significantly correlated with plasma thiol and disulfide redox measures. ConclusionsAdherence to the Mediterranean diet was significantly associated with a favorable plasma thiol/disulfide redox profile, independent of BMI, in a generally healthy working adult population. Although longitudinal studies are warranted, these findings contribute to the feasibility of targeting a Mediterranean diet to improve plasma redox status.

Higher Mediterranean Diet Quality Score and Lower BMI are Associated with a Less Oxidized Plasma Glutathione and Cysteine Redox Status in Adults

BackgroundBoth plasma redox and diet quality are associated with chronic disease risk, although the extent to which diet quality influences plasma redox status is unknown. We aimed to investigate the relationships of diet quality and obesity with systemic thiol/disulfide redox status.MethodsWe performed a cross‐sectional study of 721 working adults in Atlanta, Georgia. Diet quality was assessed by diet quality scores of habitual dietary intake derived from Block food frequency questionnaires [the Alternative Healthy Eating Index (AHEI), the Dietary Approaches to Stop Hypertension (DASH) Score, and the Mediterranean Diet Score (MDS)]. We measured plasma glutathione (GSH), cysteine (Cys), and their respective oxidized forms and redox potentials to assess redox status. Linear regression modeling was performed to assess relationships, independently of body mass index (BMI) and other confounders.ResultsThe MDS was positively associated with plasma GSH (β=0.03, p&lt;0.05) and negatively associated with the cystine/GSH ratio (β=−0.03, p&lt;0.05) in multivariate models. Individual Mediterranean diet components (dairy, vegetables, fish, and monounsaturated fat) were also significantly associated with plasma redox outcomes (p&lt;0.05). AHEI and DASH indexes and other dietary factors were not significantly associated with plasma redox outcomes. Obesity status was significantly associated with more oxidized plasma redox markers and several dietary factors, including diet quality indexes and sulfur amino acid intake (p&lt;0.05).ConclusionAdherence to a Mediterranean diet was associated with less oxidizing plasma thiol/disulfide redox systems, while obesity was associated with a more oxidizing state. These findings contribute to the feasibility of targeting diet to improve plasma redox status.Support or Funding InformationSupported by Emory University and the National Institutes of Health (UL1TR000454, K24 DK096574, K01 DK102851).

A novel mechanism of functional cooperativity regulation by thiol redox status in a dimeric inorganic pyrophosphatase

BackgroundInorganic PPases are essential metal-dependent enzymes that convert pyrophosphate into orthophosphate. This reaction is quite exergonic and provides a thermodynamic advantage for many ATP-driven biosynthetic reactions. We have previously demonstrated that cytosolic PPase from R. microplus embryos is an atypical Family I PPase. Here, we explored the functional role of the cysteine residues located at the homodimer interface, its redox sensitivity, as well as structural and kinetic parameters related to thiol redox status. MethodsIn this work, we used prokaryotic expression system for recombinant protein overexpression, biochemical approaches to assess kinetic parameters, ticks embryos and computational approaches to analyze and predict critical amino acids as well as physicochemical properties at the homodimer interface. ResultsCysteine 339, located at the homodimer interface, was found to play an important role in stabilizing a functional cooperativity between the two catalytic sites, as indicated by kinetics and Hill coefficient analyses of the WT-rBmPPase. WT-rBmPPase activity was up-regulated by physiological antioxidant molecules such as reduced glutathione and ascorbic acid. On the other hand, hydrogen peroxide at physiological concentrations decreased the affinity of WT-rBmPPase for its substrate (PPi), probably by inducing disulfide bridge formation. ConclusionsOur results provide a new angle in understanding redox control by disulfide bonds formation in enzymes from hematophagous arthropods. The reversibility of the down-regulation is dependent on hydrophobic interactions at the dimer interface. General significanceThis study is the first report on a soluble PPase where dimeric cooperativity is regulated by a redox mechanism, according to cysteine redox status.

A simple isotopic labeling method to study cysteine oxidation in Alzheimer's disease: oxidized cysteine-selective dimethylation (OxcysDML).

Cysteine is widely involved in redox signaling pathways through a number of reversible and irreversible modifications. Reversible modifications (e.g., S-glutathionylation, S-nitrosylation, disulfide bonds, and sulfenic acid) are used to protect proteins from oxidative attack and maintain cellular homeostasis, while irreversible oxidations (e.g., sulfinic acid and sulfonic acid) serve as hallmarks of oxidative stress. Proteomic analysis of cysteine-enriched peptides coupled with reduction of oxidized thiols can be used to measure the oxidation states of cysteine, which is helpful for elucidating the role that oxidative stress plays in biology and disease. As an extension of our previously reported cysDML method, we have developed oxidized cysteine-selective dimethylation (OxcysDML), to investigate the site-specific total oxidation of cysteine residues in biologically relevant samples. OxcysDML employs (1) blocking of free thiols by a cysteine-reactive reagent, (2) enrichment of peptides containing reversibly oxidized cysteine by a solid phase resin, and (3) isotopic labeling of peptide amino groups to quantify cysteine modifications arising from different biological conditions. On-resin enrichment and labeling minimizes sample handing time and improves efficiency in comparison with other redox proteomic methods. OxcysDML is also inexpensive and flexible, as it can accommodate the exploration of various cysteine modifications. Here, we applied the method to liver tissues from a late-stage Alzheimer's disease (AD) mouse model and wild-type (WT) controls. Because we have previously characterized this proteome using the cysDML approach, we are able here to probe deeper into the redox status of cysteine in AD. OxcysDML identified 1129 cysteine sites (from 527 proteins), among which 828 cysteine sites underwent oxidative modifications. Nineteen oxidized cysteine sites had significant alteration levels in AD and represent proteins involved in metabolic processes. Overall, we have demonstrated OxcysDML as a simple, rapid, robust, and inexpensive redox proteomic approach that is useful for gaining deeper insight into the proteome of AD.

Identification of thioredoxin targets in guard cell enriched epidermal peels using cysTMT proteomics

Thioredoxins (Trx) play central roles in cellular redox regulation. Although hundreds of Trx targets have been identified using different approaches, the capture of targets in a quantitative and efficient manner is challenging. Here we report a high-throughput method using cysteine reactive tandem mass tag (cysTMT) labeling followed by liquid chromatography (LC)–mass spectrometry (MS) to screen for Trx targets. Compared to existing methods, this approach allows for i) three replicates of pairwise comparison in a single LC–MS run to reduce run-to-run variation; ii) efficient enrichment of cysteine-containing peptides that requires low protein input; and iii) accurate quantification of the cysteine redox status and localization of the Trx targeted cysteine residues. Application of this method in guard cell-enriched epidermal peels from Brassica napus revealed 80 Trx h targets involved in a broad range of processes, including photosynthesis, stress response, metabolism and cell signaling. The adaption of this protocol in other systems will greatly improve our understanding of the Trx function in regulating cellular redox homeostasis. Biological significanceRedox homeostasis is tightly regulated for proper cellular activities. Specific protein–protein interactions between redox active molecules such as thioredoxin (Trx) and target proteins constitute the basis for redox-regulated biological processes. The use of cysTMT quantitative proteomics for studying Trx reactions enabled identification of potential Trx targets that provide important insights into the redox regulation in guard cells, a specialized plant cell type responsible for sensing of environmental signals, gas exchange and plant productivity.

Effect of N-Acetyl-L-Cysteine on Cysteine Redox Status in Patients with Thrombotic Thrombocytopenic Purpura: Protein Disulfide Bound Cysteine As a Biomarker of Oxidative Stress

N-Acetyl-L-Cysteine (NAC), an antioxidant drug, has been used to treat many diseases associated with oxidative stress. Nevertheless, its molecular mechanism of action in vivo remains to be understood. In an ongoing pilot study of NAC to treat thrombotic thrombocytopenic purpura (TTP), we found that NAC treatment following plasma exchange was safe in two TTP patients accompanying with rapid recovery. To explore whether the effectiveness of NAC is linked to plasma redox status, we developed a mass spectrometry based approach to measure cysteine (Cys) and Cys-containing disulfides including protein-bound cysteine (p-ss-Cys) in plasma, and measured these molecular species in plasma from patients with TTP before or after treatment with NAC. We propose that effect of NAC on p-ss-Cys may serve as an indicator for NAC action on redox sensitive proteins in plasma.Methods: Free thiols in plasma were blocked by N-ethylamaleimide (NEM), which also stops further disulfide exchange. NEM blocked thiols and small-molecule disulfides were extracted by methanol with isotopically-labeled internal standards and analyzed by liquid chromatography-tandem mass spectrometry with multiple reaction monitoring (LC-MS/MS-MRM). To determine total Cys including p-ss-Cys, we revaluated a parallel aliquot of plasma first reducing all disulfide-bound Cys with dithiothreitol (DTT) and then blocking the thiols with NEM before methanol extraction. Blood samples from TTP patients and normal donors were collected under protocols approved by IRB. Citrated Blood was collected daily from two patients with relapsed TTP before, during, andafter NAC treatment.Results: We first compared the concentrations of free-thiol Cys and Cys-containing disulfides in the plasma from two TTP patients before plasma exchange to plasma from normal donors. The concentrations of free-thiol Cys were in the low µM range and there were no significant differences between normal donor and TTP patients. However, the concentration of p-ss-Cys was greatly increased in the plasma from both TTP patients (337±33 µM) compared to normal heathy controls (181±27 µM, n=15). After plasma exchange prior to NAC infusion, the concentrations of p-ss-Cys in plasma from both patients decreased to the normal range. During a 4-day NAC treatment at doses of 300 mg/kg/day, p-ss-Cys further decreased to less than 80 µM in both TTP patients. The decreased p-ss-Cys was associated with increased the total free thiol concentration in patient plasma. Interestingly, we observed the lowest level of p-ss-Cys after the 3rd day of NAC treatment in patient 1, while it reached the lowest concentration after only one day of NAC treatment in patient 2. The different response to NAC treatment in the two patients may reflect that the patients are under different extent of oxidative stress.Conclusions: Our results suggest that TTP is associated with a high level of oxidative stress, as determined by accumulation of protein-bound Cys in plasma. NAC effectively reduces the disulfides that attach Cys to proteins, an effect that can be monitored by mass spectrometry. These results indicate that protein-ss-Cys can serve as a plasma biomarker for oxidative stress and effectiveness of antioxidant therapy. DisclosuresNo relevant conflicts of interest to declare.

Hyperglycemia and redox status regulate RUNX2 DNA-binding and an angiogenic phenotype in endothelial cells

Angiogenesis is regulated by hyperglycemic conditions, which can induce cellular stress responses, reactive oxygen species (ROS), and anti-oxidant defenses that modulate intracellular signaling to prevent oxidative damage. The RUNX2 DNA-binding transcription factor is activated by a glucose-mediated intracellular pathway, plays an important role in endothelial cell (EC) function and angiogenesis, and is a target of oxidative stress. RUNX2 DNA-binding and EC differentiation in response to glucose were conserved in ECs from different tissues and inhibited by hyperglycemia, which stimulated ROS production through the aldose reductase glucose-utilization pathway. Furthermore, the redox status of cysteine and methionine residues regulated RUNX2 DNA-binding and reversal of oxidative inhibition was consistent with an endogenous Methionine sulfoxide reductase-A (MsrA) activity. Low molecular weight MsrA substrates and sulfoxide scavengers were potent inhibitors of RUNX2 DNA binding in the absence of oxidative stress, but acted as antioxidants to increase DNA binding in the presence of oxidants. MsrA was associated with RUNX2:DNA complexes, as measured by a sensitive, quantitative DNA-binding ELISA. The related RUNX2 protein family member, RUNX1, which contains an identical DNA-binding domain, was a catalytic substrate of recombinant MsrA. These findings define novel redox pathways involving aldose reductase and MsrA that regulate RUNX2 transcription factor activity and biological function in ECs. Targeting of these pathways could result in more effective strategies to alleviate the vascular dysfunction associated with diabetes or cancer.

Chasing cysteine oxidative modifications: proteomic tools for characterizing cysteine redox status.

Redox-proteomics involves the large scale analysis of oxidative protein post-translational modifications. In particular, cysteine residues have become the subject of intensifying research interest because of their redox-reactive thiol side chain. Certain reactive cysteine residues can function as redox-switches, which sense changes in the local redox-environment by flipping between the reduced and oxidized state. Depending on the reactive oxygen or nitrogen species, cysteine residues can receive one of several oxidative modifications, each with the potential to confer a functional effect. Modification of these redox-switches has been found to play an important role in oxidative-signaling in the cardiovascular system and elsewhere. Due to the labile and dynamic nature of these modifications, several targeted approaches have been developed to enrich, identify and characterize the status of these critical residues. Here, we review the various proteomic strategies and limitations for the large scale analysis of the different oxidative cysteine modifications.

Identification and Characterization of Buried Unpaired Cysteines in a Recombinant Monoclonal IgG1 Antibody

The heterogeneity in therapeutic antibodies arising from buried unpaired cysteines has not been well studied. This paper describes the characterization of two unpaired cysteines in a recombinant humanized IgG1 monoclonal antibody (referred to as mAb A). The reversed-phase high-performance liquid chromatography (RP-HPLC) analysis of mAb A samples showed three distinct peaks, indicating the presence of three species. The heterogeneities observed in the RP-HPLC have been determined to arise from unpaired cysteines (Cys-22 and Cys-96) that are buried in the V(H) domain. The Fab containing free thiols (referred to as "free-thiol Fab") and the Fab containing the disulfide (referred to as "intact Fab") of mAb A were generated through limited Lys-C digestion and purified with an ion exchange chromatography method. The binding of free-thiol Fab and intact Fab to its antigen was measured in a cell-based binding assay and an enzyme linked immunosorbent assay. The unpaired cysteines in the Fab of mAb A were found to have no significant impact on the binding to its target. Consistent with these Fab binding data, the enriched intact mAb A containing free thiols was determined to be fully active in a potency assay. The data reported here demonstrate that the redox status of cysteines is potentially a major source of heterogeneity for an antibody.

Redox Status of Main Urinary Sulfur Amino Acids Evaluation by Liquid Chromatography

Thiol redox status, defined as reduced-to-oxidized ratio, of main urinary aminothiols has been measured. Both forms of thiols were determined by liquid chromatography with ultraviolet detection. The analytes were derivatized with thiol-specific ultraviolet labeling reagent, 2-chloro-1-methylquinolinium tetrafluoroborate, separated chromatographically and detected at 355 nm. Oxidized forms were converted to their thiol counterparts by reductive cleavage with tris(2-carboxyethyl)phosphine hydrochloride prior to derivatization step. Redox status of cysteine and cysteinylglycine was determined in urine of 45 healthy volunteers distributed for age. There were no significant differences in cysteine redox status depending on the age (P > 0.05), whereas cysteinylglycine redox status was significantly lower in children than in adults (P 0.05) between age groups.

GSH is required to maintain gut intracellular cysteine redox status after intestinal surgery in rats

BACKGROUND Glutathione (GSH) and cysteine (Cys) are major low molecular thiols that maintain cellular and plasma redox balance. Available data suggest that GSH may also be important in maintaining Cys homeostasis in vivo. Using buthionine sulfoximine (BSO), a specific rate-limiting inhibitor of GSH synthesis, to deplete tissue and plasma GSH, we investigated regulation of Cys concentration and redox status by GSH in a rat model of intestinal surgery. METHODS Adult rats underwent laparotomy, jejunal transection and anastomosis, with (n = 6) or without (n = 7) administration of BSO from day 4 until animal sacrifice 7 days after operation. BSO-treated and control rats were pair-fed. We measured mucosal and plasma thiol status [GSH, glutathione disulfide (GSSG), Cys and cystine (CySS) levels and redox potential (Eh)] and gut free malondialdehyde (MDA), an index of lipid peroxidation. RESULTS BSO markedly decreased ileal, colonic and plasma GSH and GSSG concentrations. Although Cys is a substrate for GSH synthesis (blocked by BSO), mucosal Cys was paradoxically depleted in BSO-treated rats [−32% in ileum (P = 0.06), −68% (P < 0.05) in colon], suggesting impaired cellular cyst(e)ine uptake. Such thiol depletion resulted in oxidative stress, indicated by oxidation of ileal and colonic GSH/GSSG and Cys/CySS Eh, and increased MDA content. In contrast to decreased gut mucosal Cys and CySS, BSO induced a concomitant increase in plasma Cys (+98%, P < 0.05) and CySS (+28%, P < 0.05), indicating impaired tissue extraction from blood. CONCLUSIONS GSH is required to maintain Cys redox status in gut mucosa in rats subjected to major abdominal surgery. Gut mucosal oxidative stress induced by GSH depletion may be exacerbated by impaired GSH dependent Cys uptake from blood in this model. (NIH: R01 DK55850)

S-Thiolation mimicry: Quantitative and kinetic analysis of redox status of protein cysteines by glutathione-affinity chromatography

S-Glutathionylation is emerging as a novel regulatory and adoptive mechanism by which glutathione (GSH or GSSG) conjugation can modify functionally important reactive cysteines in redox-sensitive proteins. The dynamics of generation and reversal of this modification in cells is poorly understood. This study describes the ability and applicability of GSH- and GSSG-affinity matrices to quantitatively bind proteins which harbor reactive cysteines and undergo glutathionylation. We showed that purified proteins, known to be modified by S-thiolation, bind to these matrices, are selectively eluted by dithiothreitol and rapidly incorporate biotin-labeled GSH or GSSG in vitro. Chromatography of extracts from tumor cells that had been treated with oxidants (diamide, H 2O 2, tert-butyl hydroperoxide) on GSH–Sepharose showed the specific binding of many proteins, whose levels increased transiently (2- to 6-fold) soon after treatments. However, when these cells were post-incubated in drug/oxidant-free media, protein binding decreased gradually to control levels over 3–12 h, thereby demonstrating the central role of cysteine redox status in the binding. Immunoblotting of eluates from GSH–Sepharose showed the presence of known (actin, ubiquitin-activating enzyme E1, NF-κB, and proteasome) and putative (p53, glutathione- S-transferase P1) targets for glutathionation. After oxidant withdrawal, many of these proteins displayed unique kinetics in their loss of binding to GSH-matrix, reflecting their differential abilities to recover from cysteine redox changes in cellular milieu. Further, we correlated the kinetics of S-thiolation susceptibility of the proteasome and ubiquitin-E1 proteins with altered levels of protein ubiquitination in H 2O 2-treated cells. Our study reveals the hitherto underutilized ability of glutathione matrices for analyzing the kinetics of cysteine redox in cellular proteins and allows easy identification of S-thiolatable proteins.

Quality control in protein biogenesis: thiol-mediated retention monitors the redox state of proteins in the endoplasmic reticulum.

There is accumulating evidence that proteins can be retained in the endoplasmic reticulum by a mechanism that is believed to monitor the oxidation status of one or more cysteines in their sequences. For example, a single cysteine residue critical for retention of secretory IgM assembly intermediates has been mapped to the C-terminal cysteine, Cys575, of the secretory mu chain. Little is known concerning the mechanism responsible for this system of quality control, which has been termed thiolmediated retention. In particular, it is not known if the mechanism monitors the redox state of the important cysteine residue in the secretory mu protein itself or within the context of higher-order IgM complexes. To address this question, we evaluated the fidelity of retention of secretory IgM and determined the redox status of cysteines in secretory mu proteins in polymers and polymer intermediates at various stages of maturation. We demonstrate that all secreting B cells and B cell lines secrete assembly intermediates in addition to completed, covalent pentameric and hexameric IgM polymers. A fraction of assembly intermediates exit the endoplasmic reticulum as individual components, mature through the Golgi without undergoing further assembly, and most, if not all, are secreted. While the majority of IgM assembly intermediates have exposed thiols and are contained within the endoplasmic reticulum where they can be utilized for oligomerization, maturing assembly intermediates found in the Golgi and extracellular space are completely oxidized. Thus, while the retention of unpolymerized IgM is highly efficient, the retention system lacks the ability to distinguish fully oxidized assembly intermediates from fully oxidized completed polymers. The molecular mechanisms that may contribute to this aspect of IgM biogenesis and their implications for the concept of thiolmediated retention are discussed.

Determination of the in vivo redox status of cysteine, cysteinylglycine, homocysteine, and glutathione in human plasma

An assay that measures the reduced, oxidized, and protein-bound forms of cysteine, cysteinylglycine, homocysteine, and glutathione in human plasma is described. Oxidized and protein-bound thiols are converted to their reduced counterparts by the use of NaBH 4, and, following derivatization with monobromobimane (mBrB), the thiol-bimane adducts are quantified by reversed-phase ion-pair liquid chromatography and fluorescence detection. The presence of 50 μ m dithioerythritol provides linearity of the standard curves at very low thiol concentrations. Selective determination of the oxidized forms was accomplished by blocking free sulfhydryl groups with N-ethylmaleimide (NEM) and excess NEM is inactivated by the subsequent addition of NaBH 4. The reduced forms of the thiols in plasma were trapped with minimal oxidation by derivatizing blood samples at the time of collection. This was attained by drawing blood directly into tubes containing isotonic solutions of mBrB or NEM. The assay is sufficiently sensitive (<2 pmol) to detect the various forms of the four thiol compounds in human plasma. plasma. The analytical recovery of cysteine, cysteinylglycine, homocysteine, and glutathione was close to 100%, and the within-day precision corresponded to a coefficient of variation of 7, 8, 6, and 7%, respectively. The assay has been used to determine the various forms of the four thiol compounds in human plasma.