Protein-bound Cysteine Research Articles

A High-Throughput Absolute Quantification of Protein-Bound Sulfur Amino Acids from Model and Crop Plant Seeds.

In this procedure, we describe a high-throughput absolute quantification protocol for the protein-bound sulfur amino acids, cysteine (Cys) and methionine (Met), from plant seeds. This procedure consists of performic acid oxidation that transforms bound Cys into cysteic acid (CysA) and bound Met into methionine sulfone (MetS) followed by acid hydrolysis. The absolute quantification step is performed by multiple reaction monitoring tandem mass spectrometry (LC-MS/MS). The approach facilitates the analysis of a few hundred samples per week by using a 96-well plate extraction setup. Importantly, the method uses only ∼4 mg of tissue per sample and uses the common acid hydrolysis protocol, followed by water extraction that includes DL-Ser-d3 and L-Met-d3 as internal standards to enable the quantification of the absolute levels of the protein-bound Cys and Met with high precision, accuracy, and reproducibility. The protocol described herein has been optimized for seed samples from Arabidopsis thaliana, Glycine max, and Zea mays but could be applied to other plant tissues. © 2023 Wiley Periodicals LLC. Basic Protocol: Analysis of protein-bound cysteine and methionine from seeds.

New Aminobiphenylcysteine Derivatives in Globin and Urine of Rats Dosed with 4-Aminobiphenyl, a Tobacco Smoke Carcinogen.

The 4-biphenylnitrenium ion (BPN), a reactive metabolic intermediate of the tobacco smoke carcinogen 4-aminobiphenyl (4-ABP), can react with nucleophilic sulfanyl groups in glutathione (GSH) as well as in proteins. The main site of attack of these S-nucleophiles was predicted using simple orientational rules of aromatic nucleophilic substitution. Thereafter, a series of presumptive 4-ABP metabolites and adducts with cysteine were synthesized, namely, S-(4-amino-3-biphenyl)cysteine (ABPC), N-acetyl-S-(4-amino-3-biphenyl)cysteine (4-amino-3-biphenylmercapturic acid, ABPMA), S-(4-acetamido-3-biphenyl)cysteine (AcABPC), and N-acetyl-S-(4-acetamido-3-biphenyl)cysteine (4-acetamido-3-biphenylmercapturic acid, AcABPMA). Then, globin and urine of rats dosed with a single ip dose of 4-ABP (27 mg/kg b.w.) was analyzed by HPLC-ESI-MS2. ABPC was identified in acid-hydrolyzed globin at levels of 3.52 ± 0.50, 2.74 ± 0.51, and 1.25 ± 0.12 nmol/g globin (mean ± S.D.; n = 6) on days 1, 3, and 8 after dosing, respectively. In the urine collected on day 1 (0-24 h) after dosing, excretion of ABPMA, AcABPMA, and AcABPC amounted to 1.97 ± 0.88, 3.09 ± 0.75, and 3.69 ± 1.49 nmol/kg b.w. (mean ± S.D.; n = 6), respectively. On day 2, excretion of the metabolites decreased by one order of magnitude followed by a slower decrease on day 8. Regarding the possible formation of AcABPC from ABPC, N-acetylation of the amino group at the biphenyl moiety prior to that at cysteine appears to be very unlikely. Thus, the structure of AcABPC indicates the involvement of N-acetyl-4-biphenylnitrenium ion (AcBPN) and/or its reactive ester precursors in in vivo reactions with GSH and protein-bound cysteine. ABPC in globin might become an alternative biomarker of the dose of toxicologically relevant metabolic intermediates of 4-ABP.

Using tissue cysteine to predict the trophic transfer of methylmercury and selenium in lake food webs

The biomagnification of toxic methylmercury (MeHg) and selenium (Se) through aquatic food webs using nitrogen stable isotopes (δ15N) varies among ecosystems but underlying mechanisms are yet unexplained. Given the strong links between MeHg and thiol-containing amino acids and proteins containing selenocysteine, our hypothesis was that cysteine content is a better predictor of MeHg and Se transfer through lake food webs than δ15N. Food web samples were collected from six lakes in Kejimkujik National Park, Nova Scotia, Canada, and the regression slopes of log MeHg or Se versus protein-bound cysteine or bulk δ15N were compared. Across all six lakes, MeHg varied by a factor of 10 among taxa and was significantly and positively related to both cysteine (R2 = 0.65–0.80, p < 0.001) and δ15N (R2 = 0.88–0.94, p < 0.001), with no among-system differences in these slopes. In contrast, total Se concentrations varied by less than a factor of 2 among taxa in four lakes and were significantly related to cysteine in only two food webs (R2 = 0.20 & 0.37, p = 0.014 & < 0.001); however, δ15N was not a predictor of Se in any lake (p = 0.052–0.777). Overall, these novel results indicate that cysteine content predicts MeHg, and sometimes Se, across trophic levels, providing a potential mechanism for among-system differences in their biomagnification.

Overexpression of ATP sulfurylase improves the sulfur amino acid content, enhances the accumulation of Bowman–Birk protease inhibitor and suppresses the accumulation of the β-subunit of β-conglycinin in soybean seeds

ATP sulfurylase, an enzyme which catalyzes the conversion of sulfate to adenosine 5′-phosphosulfate (APS), plays a significant role in controlling sulfur metabolism in plants. In this study, we have expressed soybean plastid ATP sulfurylase isoform 1 in transgenic soybean without its transit peptide under the control of the 35S CaMV promoter. Subcellular fractionation and immunoblot analysis revealed that ATP sulfurylase isoform 1 was predominantly expressed in the cell cytoplasm. Compared with that of untransformed plants, the ATP sulfurylase activity was about 2.5-fold higher in developing seeds. High-resolution 2-D gel electrophoresis and immunoblot analyses revealed that transgenic soybean seeds overexpressing ATP sulfurylase accumulated very low levels of the β-subunit of β-conglycinin. In contrast, the accumulation of the cysteine-rich Bowman–Birk protease inhibitor was several fold higher in transgenic soybean plants when compared to the non-transgenic wild-type seeds. The overall protein content of the transgenic seeds was lowered by about 3% when compared to the wild-type seeds. Metabolite profiling by LC–MS and GC–MS quantified 124 seed metabolites out of which 84 were present in higher amounts and 40 were present in lower amounts in ATP sulfurylase overexpressing seeds compared to the wild-type seeds. Sulfate, cysteine, and some sulfur-containing secondary metabolites accumulated in higher amounts in ATP sulfurylase transgenic seeds. Additionally, ATP sulfurylase overexpressing seeds contained significantly higher amounts of phospholipids, lysophospholipids, diacylglycerols, sterols, and sulfolipids. Importantly, over expression of ATP sulfurylase resulted in 37–52% and 15–19% increases in the protein-bound cysteine and methionine content of transgenic seeds, respectively. Our results demonstrate that manipulating the expression levels of key sulfur assimilatory enzymes could be exploited to improve the nutritive value of soybean seeds.

Cysteine Disulfides (Cys-ss-X) as Sensitive Plasma Biomarkers of Oxidative Stress

We developed a high-throughput mass spectrometry–based method to simultaneously quantify numerous small-molecule thiols and disulfides in blood plasma. Application of this assay to analyze plasma from patients with known oxidative stress (sickle cell disease and sepsis) and from a patient with sickle cell disease treated with the antioxidant N-acetylcysteine suggests that cysteine disulfides, in particular protein-bound cysteine, serve as sensitive plasma biomarkers for the extent of oxidative stress and effectiveness of antioxidant treatment.

Quantitative determination of polysulfide in albumins, plasma proteins and biological fluid samples using a novel combined assays approach

Hydrogen sulfide (H2S) signaling involves polysulfide (RSSnSR′) formation on various proteins. However, the current lack of sensitive polysulfide detection assays poses methodological challenges for understanding sulfane sulfur homeostasis and signaling. We developed a novel combined assay by modifying Sulfide Antioxidant Buffer (SAOB) to produce an “Elimination Method of Sulfide from Polysulfide” (EMSP) treatment solution that liberates sulfide, followed with methylene blue (MB) sulfide detection assay. The combined EMSP-MB sulfide detection assay performed on low molecular weight sulfur species showed that sulfide was produced from trisulfide compounds such as glutathione trisulfide and diallyl trisulfide, but not from the thiol compounds such as cysteine, cystine and glutathione. In the case of plasma proteins, this novel combined detection assay revealed that approximately 14.7, 1.7, 3.9, 3.7 sulfide mol/mol released from human serum albumin, α1-anti-trypsin, α1-acid glycoprotein and ovalbumin, respectively, suggesting that serum albumin is a major pool of polysulfide in human blood circulation. Taken together with the results of albumins of different species, the liberated sulfide has a good correlation with cysteine instead of methionine, indicating the site of incorporation of polysulfide is cysteine. With this novel sulfide detention assay, approximately 8,000, 120 and 1100 μM of polysulfide concentrations was quantitated in human healthy plasma, saliva and tear, respectively. Our promising polysulfide specific detection assay can be a very important tool because quantitative determination of polysulfide sheds light on the functional consequence of protein-bound cysteine polysulfide and expands the research area of reactive oxygen to reactive polysulfide species.

A Pilot Study of High-Dose N-Acetylcysteine Infusion in Patients with Sickle Cell Disease

▪We have completed a clinical trial evaluating the safety of N-acetylcysteine (NAC) infusion in adult patients with sickle cell disease (SCD) at disease baseline (NCT01800526). This trial was inspired by the hypothesis that von Willebrand factor (VWF) has an important role in the pathophysiology of vaso-occlusion and hemolysis in SCD and that NAC can reduce signs and symptoms of the disease by reducing the activity of VWF. A secondary hypothesis was that NAC could also act by reversing or preventing some of the oxidative changes associated with SCD.VWF is stored in the Weibel-Palade bodies of endothelial cells and α-granules of platelets and released from endothelial cells upon activation and mediates the attachment of platelets to the vessel wall, and secondarily, of erythrocytes and leukocytes. We previously showed that VWF levels and adhesive activity in SCD plasma were elevated and that total active VWF correlated with the extent of hemolysis (determined by plasma LDH levels), suggesting that VWF participates in the pathophysiology of SCD (Chen et al, Blood. 2011; 117:3680‐3). In another study, we showed that NAC was able to reduce VWF multimer size and platelet-binding activity ex vivo in human plasma and in vivo in mice (Chen et al, JCI, 2011, 121:522).In this clinical trial, we enrolled 5 subjects at disease baseline, 4 with SCD and 1 with sickle trait. All subjects received two high-dose NAC infusions: initially 150 mg/kg and 4 weeks later 300 mg/kg. NAC infusion was administered in subjects 1 and 2 as a bolus infusion of half of the dose in the 1sthour and the remaining half in 7 hr. Subjects 3-5 received NAC as constant infusion for 8 hr. Blood was collected immediately before infusion, during infusion (at 1 and 4 hr), immediately after infusion (8 hr), and 24 and 72 hr after infusion. We examined blood for red blood cell (RBC) fragments, dense cells, platelet activation status, platelet-monocyte complexes and the concentrations of reduced and oxidized glutathione. We also examined plasma for VWF concentration and multimer distribution, ADAMTS13 antigen and activity, and evaluated plasma redox status by measuring the concentrations of NAC and cysteine in their reduced, oxidized, and mixed disulfide forms, as well as the concentration of protein-bound cysteine. The small molecule thiols were measured by mass spectrometry.Results:1) All subjects tolerated NAC infusion well, except that subjects 1 and 2 experienced pruritus during the bolus infusion at the 300 mg/kg dose. We therefore changed the protocol to deliver the drug over 8 hr by continuous infusion for subjects 3 through 5. 2) During NAC infusion, the percentage of dense cells and concentration of RBC fragments decreased rapidly (average decrease: low dose 44%; high dose 31%) and the change persisted up to 72 hr. 3) Platelet activation also decreased, as determined by reduced P-selectin expression (low dose, 7.7 ± 0.7% to 5.0 ± 1.9%; high dose, 17.8 ± 0.07% to 7.3% ± 0.4), PAC-1 binding (evaluates activated αΙΙbβ3), and formation of platelet-monocyte complexes (monocytes with attached platelets: low dose, 52 ± 16% to 27 ± 12%; high dose, 77 ± 12% to 50 ± 17%). 4) The highest molecular weight VWF multimers transiently disappeared during the NAC infusion and recovered by 24 hr. There were no appreciable changes in VWF antigen or ADAMTS13 antigen or activity with NAC infusion. 5) With NAC infusion, the concentrations of Cys and NAC increased in plasma, both total and reduced forms, with a concomitant decrease in protein-bound Cys in the one patient studied (free thiol Cys increased 23 fold with the high dose infusion at 8 hr; protein-bound Cys decreased 87%). Similarly, the concentrations of reduced and oxidized glutathione increased in whole blood.Summary: NAC infusion in SCD patients at disease baseline appears safe and is well tolerated. NAC infusion decreases RBC fragments, dense cells, and the size of large VWF multimers, and inhibits platelet activation and formation of platelet-monocyte complexes. In addition, NAC infusion increases the total and free-thiol concentrations of cysteine and glutathione in blood, relieving oxidative stress in SCD. Our data suggest that NAC may be of benefit for SCD patients during vaso-occlusive crisis through a variety of mechanisms, which include improvements in red cell parameters, decreased platelet adhesion, reduced adhesive activity of VWF, and protection against oxidative damage. DisclosuresNo relevant conflicts of interest to declare.

Comprehensively Characterizing the Thioredoxin Interactome In Vivo Highlights the Central Role Played by This Ubiquitous Oxidoreductase in Redox Control

Thioredoxin (Trx) is a ubiquitous oxidoreductase maintaining protein-bound cysteine residues in the reduced thiol state. Here, we combined a well-established method to trap Trx substrates with the power of bacterial genetics to comprehensively characterize the in vivo Trx redox interactome in the model bacterium Escherichia coli Using strains engineered to optimize trapping, we report the identification of a total 268 Trx substrates, including 201 that had never been reported to depend on Trx for reduction. The newly identified Trx substrates are involved in a variety of cellular processes, ranging from energy metabolism to amino acid synthesis and transcription. The interaction between Trx and two of its newly identified substrates, a protein required for the import of most carbohydrates, PtsI, and the bacterial actin homolog MreB was studied in detail. We provide direct evidence that PtsI and MreB contain cysteine residues that are susceptible to oxidation and that participate in the formation of an intermolecular disulfide with Trx. By considerably expanding the number of Trx targets, our work highlights the role played by this major oxidoreductase in a variety of cellular processes. Moreover, as the dependence on Trx for reduction is often conserved across species, it also provides insightful information on the interactome of Trx in organisms other than E. coli.

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.

Involvement of reactive persulfides in biological bismethylmercury sulfide formation.

Bismethylmercury sulfide (MeHg)2S has been found to be a detoxified metabolite of methylmercury (MeHg) that is produced by SH-SY5Y cells and in livers of rats exposed to MeHg. (MeHg)2S could be formed through the interactions between MeHg and sulfur species such as hydrogen sulfide (H2S or HS(-)), but the origin of its sulfur has not been fully identified. We herein examined the formation of (MeHg)2S through interactions between MeHg and persulfides, polysulfides, and protein preparations. Investigations using HPLC/atomic absorption spectrophotometry and EI-MS revealed that NaHS and Na2S4 react readily with MeHg to give (MeHg)2S, and similar results were found using GSH persulfide (GSSH) formed endogenously or generated enzymatically in vitro. (MeHg)2S was also formed by incubation of MeHg with liver and heart cytosolic fractions prepared from wild-type mice but not with those from mice lacking cystathionine γ-lyase (CSE) that catalyzes the formation of cysteine persulfide. Consistent with this, (MeHg)2S was detected in a variety of tissues taken from wild-type mice intraperitoneally injected with MeHg in vivo but not in those from MeHg-injected CSE knockout mice. By separating liver fractions by column chromatography, we found numerous proteins that contain persulfides: one of the proteins was identified as being glutathione S-transferase pi 1. These results indicate that the formation of (MeHg)2S can be attributed to interactions between MeHg and endogenous free persulfide species, as well as protein-bound cysteine persulfide.

Salicylic acid and cysteine contribute to arbutin-induced alleviation of angular leaf spot disease development in cucumber

Arbutin induced suppression of angular leaf spot disease in cucumber resulting from lower populations of Pseudomonas syringae pv lachrymans in the infected tissues. This study provides insight into mechanisms that may potentially account for this effect. In the absence of the pathogen, exogenous arbutin-induced expression of PR1, the marker of salicylic acid signaling, increased the content of salicylic acid and modulated the cysteine pool. This suggested that arbutin promoted cucumber plants to a “primed” state. When challenged with the pathogen, the arbutin-treated plants showed strongly reduced infection symptoms 7 days after inoculation. At this time point, they were characterized by higher contents of free and protein-bound cysteine due to higher cysteine biosynthetic capacity related to increased activities of serine acetyltransferase and cysteine synthase when compared with plants infected without arbutin treatment. Moreover, in the arbutin-treated and infected plants the contents of free salicylic acid and its conjugates were also increased, partly owing to its biosynthesis via the phenylpropanoid pathway. We suggest that arbutin-induced abrogation of angular leaf spot disease in cucumber could be mediated by salicylic acid and cysteine-based signaling.

Involvement of ascorbate, glutathione, protein S-thiolation and salicylic acid in benzothiadiazole-inducible defence response of cucumber against Pseudomonas syringae pv lachrymans

The metabolic response of acibenzolar-S-methyl (ASM)-treated cucumber (Cucumis sativus) to Pseudomonas syringae pv lachrymans was analyzed at the local and systemic levels. Changes in ascorbate, cysteine, glutathione and salicylic acid pools were studied. The pretreatment with ASM (priming) augmented the defense response of cucumber to subsequent bacterial infection as shown by the suppression of disease development in comparison to the untreated plants. ASM changed the redox state of ascorbate, cysteine and glutathione pools, and induced accumulation of cysteine and glutathione bound to proteins. It also provoked profound accumulation of free salicylic acid and its glucosylated conjugates. It has been proposed that the coupled salicylic acid and ascorbate- and thiol-based redox signaling contributes to the activation of a primed state in ASM-treated cucumber plants. ASM-primed and non-primed plants differed with respect to the intensity and dynamics of Psl-induced changes in the pathogen-inoculated leaves and in the systemic ones, especially with respect to free and protein-bound cysteine and to salicylic acid contents. Although the physiological significance of these reactions at the local and systemic levels remains to be eluicidated, the presented results support the role of salicylic acid, ascorbate, glutathione and protein S-thiolation in defense response of cucumber against P. syringae pv lachrymans.

Transgenic soybean plants overexpressing O-acetylserine sulfhydrylase accumulate enhanced levels of cysteine and Bowman–Birk protease inhibitor in seeds

Soybeans provide an excellent source of protein in animal feed. Soybean protein quality can be enhanced by increasing the concentration of sulfur-containing amino acids. Previous attempts to increase the concentration of sulfur-containing amino acids through the expression of heterologous proteins have met with limited success. Here, we report a successful strategy to increase the cysteine content of soybean seed through the overexpression of a key sulfur assimilatory enzyme. We have generated several transgenic soybean plants that overexpress a cytosolic isoform of O-acetylserine sulfhydrylase (OASS). These transgenic soybean plants exhibit a four- to tenfold increase in OASS activity when compared with non-transformed wild-type. The OASS activity in the transgenic soybeans was significantly higher at all the stages of seed development. Unlike the non-transformed soybean plants, there was no marked decrease in the OASS activity even at later stages of seed development. Overexpression of cytosolic OASS resulted in a 58-74% increase in protein-bound cysteine levels compared with non-transformed wild-type soybean seeds. A 22-32% increase in the free cysteine levels was also observed in transgenic soybeans overexpressing OASS. Furthermore, these transgenic soybean plants showed a marked increase in the accumulation of Bowman-Birk protease inhibitor, a cysteine-rich protein. The overall increase in soybean total cysteine content (both free and protein-bound) satisfies the recommended levels required for the optimal growth of monogastric animals.

Bacterial cysteine desulfurases: versatile key players in biosynthetic pathways of sulfur-containing biofactors

Cysteine desulfurases are pyridoxal 5'-phosphate-dependent homodimeric enzymes that catalyze the conversion of L-cysteine to L-alanine and sulfane sulfur via the formation of a protein-bound cysteine persulfide intermediate on a conserved cysteine residue. The enzymes are capable of donating the persulfide sulfur atoms to a variety of biosynthetic pathways for sulfur-containing biofactors, such as iron-sulfur clusters, thiamin, transfer RNA thionucleosides, biotin, and lipoic acid. The enormous advances in biochemical and structural studies of these biosynthetic pathways over the past decades provide an opportunity for detailed understanding of the nature of the excellent sulfur transfer mechanism of cysteine desulfurases.

Mechanism for the Hydrolysis of a Sulfur-Sulfur Bond Based on the Crystal Structure of the Thiosulfohydrolase SoxB

SoxB is an essential component of the bacterial Sox sulfur oxidation pathway. SoxB contains a di-manganese(II) site and is proposed to catalyze the release of sulfate from a protein-bound cysteine S-thiosulfonate. A direct assay for SoxB activity is described. The structure of recombinant Thermus thermophilus SoxB was determined by x-ray crystallography to a resolution of 1.5 A. Structures were also determined for SoxB in complex with the substrate analogue thiosulfate and in complex with the product sulfate. A mechanistic model for SoxB is proposed based on these structures.

Crystal Structure of a Sulfur Carrier Protein Complex Found in the Cysteine Biosynthetic Pathway of Mycobacterium tuberculosis

The structure of the protein complex CysM−CysO from a new cysteine biosynthetic pathway found in the H37Rv strain of Mycobacterium tuberculosis has been determined at 1.53 Å resolution. CysM (Rv1336) is a PLP-containing β-replacement enzyme and CysO (Rv1335) is a sulfur carrier protein with a ubiquitin-like fold. CysM catalyzes the replacement of the acetyl group of O-acetylserine by CysO thiocarboxylate to generate a protein-bound cysteine that is released in a subsequent proteolysis reaction. The protein complex in the crystal structure is asymmetric with one CysO protomer binding to one end of a CysM dimer. Additionally, the structures of CysM and CysO were determined individually at 2.8 and 2.7 Å resolution, respectively. Sequence alignments with homologues and structural comparisons with CysK, a cysteine synthase that does not utilize a sulfur carrier protein, revealed high conservation of active site residues; however, residues in CysM responsible for CysO binding are not conserved. Comparison of the CysM−CysO binding interface with other sulfur carrier protein complexes revealed a similarity in secondary structural elements that contribute to complex formation in the ThiF−ThiS and MoeB−MoaD systems, despite major differences in overall folds. Comparison of CysM with and without bound CysO revealed conformational changes associated with CysO binding.

The glial Gomori-positive material is sulfane sulpfur.

The Gomori-positive glia are periventricular astrocytes with abundant cytoplasmic granular material, predominantly occupying a periventricular site in the brain. These granular inclusions are strongly stained with chrome hematoxylin in the Gomori's method as well as exhibit red autofluorescence and non-enzymatic peroxidase activity. The glial Gomori-positive material (GGPM) granules are positive in the performic acid Alcian blue method indicating the presence of protein-bound sulfur, what has been shown by our previous studies. The number of cells containing glial Gomori-positive granules dropped after administration of cyanide and increased under the influence of sulfane sulfur donor (diallyl disulfide). This suggests, that sulfur of these granules is a sulfane sulfur, possibly in the form of protein-bound cysteine persulfide. Sulfane sulfur is labile, reactive sulfur atom covalently bound to another sulfur atom. In this paper we present evidence that GGPM exhibit affinity to cyanolysis and its stainability in Gomori's method is due to the presence of protein-bound sulfane sulfur. The biological role of the Gomori-positive glia connected with protective properties of sulfane sulfur has been discussed.

Functional Characterization of a Methionine -Lyase in Arabidopsis and its Implication in an Alternative to the Reverse Trans-sulfuration Pathway

Methionine gamma-lyase (MGL) catalyzes the degradation of L-methionine to alpha-ketobutyrate, methanethiol and ammonia. The Arabidopsis (Arabidopsis thaliana) genome includes a single gene (At1g64660) encoding a protein (AtMGL) with approximately 35% identity to bacterial and protozoan MGLs. When overexpressed in Escherichia coli, AtMGL allowed growth on L-methionine as sole nitrogen source and conferred a high rate of methanethiol emission. The purified recombinant protein exhibited a spectrum typical of pyridoxal 5'-phosphate enzymes, and had high activity toward l-methionine, L-ethionine, L-homocysteine and seleno-L-methionine, but not L-cysteine. Quantitation of mRNA showed that the AtMGL gene is expressed in aerial organs and roots, and that its expression in leaves was increased 2.5-fold by growth on low sulfate medium. Emission of methanethiol from Arabidopsis plants supplied with 10 mM L-methionine was undetectable (<0.5 nmol min(-1) g(-1) FW), suggesting that AtMGL is not an important source of volatile methanethiol. Knocking out the AtMGL gene significantly increased leaf methionine content (9.2-fold) and leaf and root S-methylmethionine content (4.7- and 7-fold, respectively) under conditions of sulfate starvation, indicating that AtMGL carries a significant flux in vivo. In Arabidopsis plantlets fed L-[(35)S]methionine on a low sulfate medium, label was incorporated into protein-bound cysteine as well as methionine, but incorporation into cysteine was significantly (30%) less in the knockout mutant. These data indicate that plants possess an alternative to the reverse trans-sulfuration pathway (methionine-->homocysteine-->cystathionine-->cysteine) in which methanethiol is an intermediate.

Determination of free and total cyst(e)ine in plasma of dogs and cats

In human blood, the amino acid cysteine forms disulfide bonds with itself and with other sulfhydryl compounds in their free form and with sulfhydryls in protein. Protein-bound cysteine is lost when plasma proteins are removed before amino acid analysis. The purpose of this study was to assess the time course and extent of cyst(e)ine (cysteine + half-cystine) loss in dog and cat plasma. An equal volume of 6% sulfosalicylic acid was added to plasma aliquots at 0, 2, 4, 10, 16, 24, 36, 48, 60, and 72 hours after separation of blood cells. Tris-2-carboxyethyl-phosphine hydrochloride (TCEP - HCl), a reducing agent, was used to regenerate total plasma cyst(e)ine after 3 months of sample storage (-20 degrees C). Initial free cyst(e)ine concentrations (mean +/- SEM) were higher in canine plasma (77 +/- 4 micromol/L) than in feline plasma (37 +/- 3 micromol/L). Free plasma cyst(e)ine concentrations in dogs and cats decreased after first-order kinetics, with a half-life of 23 and 69 hours, respectively. Total plasma cysteine after TCEP - HCl treatment was similar for dogs (290 micromol/L) and cats (296 micromol/L), but the percentage of free cysteine was higher (P = .02) in dogs (27%) than in cats (13%). Over half of the cyst(e)ine, homocysteine, cysteinylglycine, and glutathione were bound in vivo to plasma proteins. These results emphasize the importance of removing plasma proteins within 1 hour after blood collection for reliable assay of free plasma cyst(e)ine.

Enhanced levels of methionine and cysteine in transgenic alfalfa (Medicago sativaL.) plants over‐expressing theArabidopsiscystathionine γ‐synthase gene

With the aim of increasing the methionine level in alfalfa (Medicago sativa L.) and thus improving its nutritional quality, we produced transgenic alfalfa plants that expressed the Arabidopsis cystathionine gamma-synthase (AtCGS), the enzyme that controls the synthesis of the first intermediate metabolite in the methionine pathway. The AtCGS cDNA was driven by the Arabidopsis rubisco small subunit promoter to obtain expression in leaves. Thirty transgenic plants were examined for the transgene protein expression, and four lines with a high expression level were selected for further work. In these lines, the contents of methionine, S-methylmethionine (SMM), and methionine incorporated into the water-soluble protein fraction increased up to 32-fold, 19-fold, and 2.2-fold, respectively, compared with that in wild-type plants. Notably, in these four transgenic lines, the levels of free cysteine (the sulphur donor for methionine synthesis), glutathione (the cysteine storage and transport form), and protein-bound cysteine increased up to 2.6-fold, 5.5-fold, and 2.3-fold, respectively, relative to that in wild-type plants. As the transgenic alfalfa plants over-expressing AtCGS had significantly higher levels of both soluble and protein-bound methionine and cysteine, they may represent a model and target system for improving the nutritional quality of forage crops.