Thiol redox-sensitive seed proteome in dormant and non-dormant hybrid genotypes of wheat

The thiol redox-sensitive and the total proteome in harvest-ripe grains of closely related genotypes of wheat (Triticum aestivum L.), with either a dormant or a non-dormant phenotype, were investigated using hybrid lines of spring wheat double haploid population segregating transgressively, to gain further insight into seed dormancy controlling events. Redox signalling by reactive oxygen species has been shown to play a role in seed dormancy alleviation. Thiol-disulfide proteins are of particular importance in the context of redox-dependent regulation as a central and flexible mechanism to control metabolic and developmental activities of the cells. Here we describe functional proteomic profiling of reversible oxidoreductive changes and characterize in vivo intrinsic reactivity of cysteine residues using thiol-specific fluorescent labelling, solubility-based protein fractionation, two-dimensional electrophoresis, and mass spectrometry analysis in conjunction with wheat EST sequence libraries. Quantitative differences between genotypes were found for 106 spots containing 64 unique proteins. Forty seven unique proteins displayed distinctive abundance pattern, and among them 31 proteins contained 78 unique redox active cysteines. Seventeen unique proteins with 19 reactive modified cysteines were found to have differential post-translational thiol redox modification. The results provide an insight into the alteration of thiol-redox profiles in proteins that function in major processes in seeds and include groups of redox- and stress-responsive, genetic information processing and cell cycle control, transport and storage proteins, enzymes of carbohydrate metabolism, proteases and their inhibitors.     

The internal Cys-207 of sorghum leaf NADP-malate dehydrogenase can form mixed disulphides with thioredoxin

The role of the internal Cys-207 of sorghum NADP-malate dehydrogenase (NADP-MDH) in the activation of the enzyme has been investigated through the examination of the ability of this residue to form mixed disulphides with thioredoxin mutated at either of its two active-site cysteines. The h-type Chlamydomonas thioredoxin was used, because it has no additional cysteines in the primary sequence besides the active-site cysteines. Both thioredoxin mutants proved equally efficient in forming mixed disulphides with an NADP-MDH devoid of its N-terminal bridge either by truncation, or by mutation of its N-terminal cysteines. They were poorly efficient with the more compact WT oxidised NADP-MDH. Upon mutation of Cys-207, no mixed disulphide could be formed, showing that this cysteine is the only one, among the four internal cysteines, which can form mixed disulphides with thioredoxin. These experiments confirm that the opening of the N-terminal disulphide loosens the interaction between subunits, making Cys-207, located at the dimer contact area, more accessible.     

Comparative proteomic analysis of cysteine oxidation in colorectal cancer patients

Oxidative stress promotes damage to cellular proteins, lipids, membranes and DNA, and plays a key role in the development of cancer. Reactive oxygen species disrupt redox homeostasis and promote tumor formation by initiating aberrant activation of signaling pathways that lead to tumorigenesis. We used shotgun proteomics to identify proteins containing oxidation-sensitive cysteines in tissue specimens from colorectal cancer patients. We then compared the patterns of cysteine oxidation in the membrane fractions between the tumor and non-tumor tissues. Using nano-UPLC-MS^E proteomics, we identified 31 proteins containing 37 oxidation-sensitive cysteines. These proteins were observed with IAM-binding cysteines in non-tumoral region more than tumoral region of CRC patients. Then using the Ingenuity pathway program, we evaluated the cellular canonical networks connecting those proteins. Within the networks, proteins with multiple connections were related with organ morphology, cellular metabolism, and various disorders. We have thus identified networks of proteins whose redox status is altered by oxidative stress, perhaps leading to changes in cellular functionality that promotes tumorigenesis.     

The disulphide isomerase DsbC cooperates with the oxidase DsbA in a DsbD-independent manner

In Escherichia coli, DsbA introduces disulphide bonds into secreted proteins. DsbA is recycled by DsbB, which generates disulphides from quinone reduction. DsbA is not known to have any proofreading activity and can form incorrect disulphides in proteins with multiple cysteines. These incorrect disulphides are thought to be corrected by a protein disulphide isomerase, DsbC, which is kept in the reduced and active configuration by DsbD. The DsbC/DsbD isomerization pathway is considered to be isolated from the DsbA/DsbB pathway. We show that the DsbC and DsbA pathways are more intimately connected than previously thought. dsbA(-)dsbC(-) mutants have a number of phenotypes not exhibited by either dsbA(-), dsbC(-) or dsbA(-)dsbD(-) mutations: they exhibit an increased permeability of the outer membrane, are resistant to the lambdoid phage Phi80, and are unable to assemble the maltoporin LamB. Using differential two-dimensional liquid chromatographic tandem mass spectrometry/mass spectrometry analysis, we estimated the abundance of about 130 secreted proteins in various dsb(-) strains. dsbA(-)dsbC(-) mutants exhibit unique changes at the protein level that are not exhibited by dsbA(-)dsbD(-) mutants. Our data indicate that DsbC can assist DsbA in a DsbD-independent manner to oxidatively fold envelope proteins. The view that DsbC's function is limited to the disulphide isomerization pathway should therefore be reinterpreted.     

Redox state of low-molecular-weight thiols and disulphides during somatic embryogenesis of salt-treated suspension cultures of Dactylis glomerata L

The tripeptide antioxidant γ-L-glutamyl-L-cysteinyl-glycine, or glutathione (GSH), serves a central role in ROS scavenging and oxidative signalling. Here, GSH, glutathione disulphide (GSSG), and other low-molecular-weight (LMW) thiols and their corresponding disulphides were studied in embryogenic suspension cultures of Dactylis glomerata L. subjected to moderate (0.085 M NaCl) or severe (0.17 M NaCl) salt stress. Total glutathione (GSH + GSSG) concentrations and redox state were associated with growth and development in control cultures and in moderately salt-stressed cultures and were affected by severe salt stress. The redox state of the cystine (CySS)/2 cysteine (Cys) redox couple was also affected by developmental stage and salt stress. The glutathione half-cell reduction potential (E(GSSG/2 GSH)) increased with the duration of culturing and peaked when somatic embryos were formed, as did the half-cell reduction potential of the CySS/2 Cys redox couple (E(CySS/2 Cys)). The most noticeable relationship between cellular redox state and developmental state was found when all LMW thiols and disulphides present were mathematically combined into a 'thiol-disulphide redox environment' (E(thiol-disulphide)), whereby reducing conditions accompanied proliferation, resulting in the formation of pro-embryogenic masses (PEMs), and oxidizing conditions accompanied differentiation, resulting in the formation of somatic embryos. The comparatively high contribution of E(CySS/2 Cys) to E(thiol-disulphide) in cultures exposed to severe salt stress suggests that Cys and CySS may be important intracellular redox regulators with a potential role in stress signalling.     

Disulphide bridges in globular proteins

Disulphide bridges in proteins of known sequence, connectivity and structure were studied to search for common features. Their distribution, topology, conformation and conservation were analysed in detail. Several general patterns emerge which to some extent dictate disulphide bridge formation. For example, there is a strong preference for shorter connections, with half-cystines separated by less than 24 residues in 49% of all disulphides. Right- and left-handed disulphides occur equally; the left-handed structures adopt one predominant conformation (symmetric χ₁ = ?60 °, χ₂ = ?80 °, χ₃ = t-90 °). Cystines are generally very well conserved, in contrast to cysteines, with a free —SH group, which mutate rapidly. If a disulphide is not conserved, both cystines are mutated. The role of disulphide bridges in globular proteins is discussed.     

Reduction of the periplasmic disulfide bond isomerase, DsbC, occurs by passage of electrons from cytoplasmic thioredoxin.

The Escherichia coli periplasmic protein DsbC is active both in vivo and in vitro as a protein disulfide isomerase. For DsbC to attack incorrectly formed disulfide bonds in substrate proteins, its two active-site cysteines should be in the reduced form. Here we present evidence that, in wild-type cells, these two cysteines are reduced. Further, we show that a pathway involving the cytoplasmic proteins thioredoxin reductase and thioredoxin and the cytoplasmic membrane protein DsbD is responsible for the reduction of these cysteines. Thus, reducing potential is passed from cytoplasmic electron donors through the cytoplasmic membrane to DsbC. This pathway does not appear to utilize the cytoplasmic glutathione-glutaredoxin pathway. The redox state of the active-site cysteines of DsbC correlates quite closely with its ability to assist in the folding of proteins with multiple disulfide bonds. Analysis of the activity of mutant forms of DsbC in which either or both of these cysteines have been altered further supports the role of DsbC as a disulfide bond isomerase.     

Transmembrane electron transfer by the membrane protein DsbD occurs via a disulfide bond cascade

The cytoplasmic membrane protein DsbD transfers electrons from the cytoplasm to the periplasm of E. coli, where its reducing power is used to maintain cysteines in certain proteins in the reduced state. We split DsbD into three structural domains, each containing two essential cysteines. Remarkably, when coexpressed, these truncated proteins restore DsbD function. Utilizing this three piece system, we were able to determine a pathway of the electrons through DsbD. Our findings strongly suggest that the pathway is based on a series of multistep redox reactions that include direct interactions between thioredoxin and DsbD, and between DsbD and its periplasmic substrates. A thioredoxin-fold domain in DsbD appears to have the novel role of intramolecular electron shuttle.     

Thioredoxin and peptide methionine sulfoxide reductase: convergence of similar structure and function in distinct structural folds.

Thioredoxin (Trx) and peptide methionine sulfoxide reductase (PMSR) are small thiol oxidoreductases implicated in antioxidant defense and redox regulation of cellular processes. Here we show that the structures of Trx and PMSR exhibit resemblance in their alphabeta core regions and that the active site cysteines in two proteins occupy equivalent positions downstream of a central beta-strand and at the N-terminus of an alpha-helix. Moreover, we identified a PMSR subfamily that contains an active site CxxC motif (two cysteines separated by two other amino acids) positioned similarly to the catalytic redox active CxxC motif in Trx. However, Trx and PMSR are characterized by distinct ancient folds that differ in both orientation of secondary structures and their patterns. Trx is a member of the Trx-fold superfamily, whereas PMSR has a unique fold not found in other proteins. The data suggest that similar structures and functions of Trx and PMSR were acquired independently during evolution and point to a general strategy of identifying new redox regulatory proteins.     

Selecting thioredoxins for disulphide proteomics: target proteomes of three thioredoxins from the cyanobacterium Synechocystis sp. PCC 6803

Searching for enzymes and other proteins which can be redox-regulated by dithiol/disulphide exchange is a rapidly expanding area of functional proteomics. Recently, several experimental approaches using thioredoxins have been developed for this purpose. Thioredoxins comprise a large family of redox-active enzymes capable of reducing protein disulphides to cysteines and of participating in a variety of processes, such as enzyme modulation, donation of reducing equivalents and signal transduction. In this study we screened the target proteomes of three different thioredoxins from the unicellular cyanobacterium Synechocystis sp. PCC 6803, using site-directed active-site cysteine-to-serine mutants of its m-, x- and y-type thioredoxins. The properties of a thioredoxin that determine the outcome of such analyses were found to be target-binding capacity, solubility and the presence of non-active-site cysteines. Thus, we explored how the choice of thioredoxin affects the target proteomes and we conclude that the m-type thioredoxin, TrxA, is by far the most useful for screening of disulphide proteomes. Furthermore, we improved the resolution of target proteins on non-reducing/reducing 2-DE, leading to the identification of 14 new potentially redox-regulated proteins in this organism. The presence of glycogen phosphorylase among the newly identified targets suggests that glycogen breakdown is redox-regulated in addition to glycogen synthesis.     

Redox state of low-molecular-weight thiols and disulphides during somatic embryogenesis of salt-treated suspension cultures of Dactylis glomerata L.

Abstract

The tripeptide antioxidant γ-L-glutamyl-L-cysteinyl-glycine, or glutathione (GSH), serves a central role in ROS scavenging and oxidative signalling. Here, GSH, glutathione disulphide (GSSG), and other low-molecular-weight (LMW) thiols and their corresponding disulphides were studied in embryogenic suspension cultures of Dactylis glomerata L. subjected to moderate (0.085 M NaCl) or severe (0.17 M NaCl) salt stress. Total glutathione (GSH + GSSG) concentrations and redox state were associated with growth and development in control cultures and in moderately salt-stressed cultures and were affected by severe salt stress. The redox state of the cystine (CySS)/2 cysteine (Cys) redox couple was also affected by developmental stage and salt stress. The glutathione half-cell reduction potential (E_{GSSG/2 GSH}) increased with the duration of culturing and peaked when somatic embryos were formed, as did the half-cell reduction potential of the CySS/2 Cys redox couple (E_{CySS/2 Cys}). The most noticeable relationship between cellular redox state and developmental state was found when all LMW thiols and disulphides present were mathematically combined into a ‘thiol–disulphide redox environment’ (E_{thiol–disulphide}), whereby reducing conditions accompanied proliferation, resulting in the formation of pro-embryogenic masses (PEMs), and oxidizing conditions accompanied differentiation, resulting in the formation of somatic embryos. The comparatively high contribution of E_{CySS/2 Cys} to E_{thiol–disulphide} in cultures exposed to severe salt stress suggests that Cys and CySS may be important intracellular redox regulators with a potential role in stress signalling.     

Reciprocal Control of the Circadian Clock and Cellular Redox State - a Critical Appraisal

Redox signalling comprises the biology of molecular signal transduction mediated by reactive oxygen (or nitrogen) species. By specific and reversible oxidation of redox-sensitive cysteines, many biological processes sense and respond to signals from the intracellular redox environment. Redox signals are therefore important regulators of cellular homeostasis. Recently, it has become apparent that the cellular redox state oscillates in vivo and in vitro, with a period of about one day (circadian). Circadian time-keeping allows cells and organisms to adapt their biology to resonate with the 24-hour cycle of day/night. The importance of this innate biological time-keeping is illustrated by the association of clock disruption with the early onset of several diseases (e.g. type II diabetes, stroke and several forms of cancer). Circadian regulation of cellular redox balance suggests potentially two distinct roles for redox signalling in relation to the cellular clock: one where it is regulated by the clock, and one where it regulates the clock. Here, we introduce the concepts of redox signalling and cellular timekeeping, and then critically appraise the evidence for the reciprocal regulation between cellular redox state and the circadian clock. We conclude there is a substantial body of evidence supporting circadian regulation of cellular redox state, but that it would be premature to conclude that the converse is also true. We therefore propose some approaches that might yield more insight into redox control of cellular timekeeping.     

5,5'-Dithio-bis(2-nitrobenzoic acid) modification of cysteine improves the crystal quality of human chloride intracellular channel protein 2

Structural studies of human chloride intracellular channel protein 2 (CLIC2) had been hampered by the problem of generating suitable crystals primarily due to the protein containing exposed cysteines. Several chemical reagents were used to react with the cysteines on CLIC2 in order to modify the redox state of the protein. We have obtained high quality crystals that diffracted to better than 2.5 Å at a home X-ray source by treating the protein with 5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB). After solving the crystal structure of CLIC2, we found that the DTNB had reacted with the Cys¹¹⁴, and made CLIC2 in a homogenous oxidized state. This study demonstrated that the DTNB modification drastically improved the crystallization of CLIC2, and it implied that this method may be useful for other proteins containing exposed cysteines in general.     

Toxicity of thiols and disulphides: Involvement of free-radical species

Sulphur is essential to life, and thiols and disulphides play essential roles in cellular biochemistry. Such compounds are also widely distributed in the food of man and his domestic animals, and they are extensively used in industry. However, many thiols and disulphides have been shown to be toxic. Aliphatic, aromatic, and heterocyclic compounds of this type are haemolytic agents in animals while aminothiols have been shown to induce many cytotoxic effects in vitro and the epidithiodioxopiperazine mycotoxin, sporidesmin, is a potent hepatotoxic agent. Structure-activity relationships among these compounds and factors which modulate their harmful effects are consistent with a toxic mechanism involving redox cycling between the thiol and the corresponding disulphide. Thiyl radicals and "active oxygen" species are formed in this process, and it is suggested that these substances are responsible for initiating the tissue damage provoked by thiols and disulphides.     

Fic and non-Fic AMPylases: protein AMPylation in metazoans

Protein AMPylation refers to the covalent attachment of an AMP moiety to the amino acid side chains of target proteins using ATP as nucleotide donor. This process is catalysed by dedicated AMP transferases, called AMPylases. Since this initial discovery, several research groups have identified AMPylation as a critical post-translational modification relevant to normal and pathological cell signalling in both bacteria and metazoans. Bacterial AMPylases are abundant enzymes that either regulate the function of endogenous bacterial proteins or are translocated into host cells to hijack host cell signalling processes. By contrast, only two classes of metazoan AMPylases have been identified so far: enzymes containing a conserved filamentation induced by cAMP (Fic) domain (Fic AMPylases), which primarily modify the ER-resident chaperone BiP, and SelO, a mitochondrial AMPylase involved in redox signalling. In this review, we compare and contrast bacterial and metazoan Fic and non-Fic AMPylases, and summarize recent technological and conceptual developments in the emerging field of AMPylation.     

SP3-Enabled Rapid and High Coverage Chemoproteomic Identification of Cell-State–Dependent Redox-Sensitive Cysteines

Proteinaceous cysteine residues act as privileged sensors of oxidative stress. As reactive oxygen and nitrogen species have been implicated in numerous pathophysiological processes, deciphering which cysteines are sensitive to oxidative modification and the specific nature of these modifications is essential to understanding protein and cellular function in health and disease. While established mass spectrometry-based proteomic platforms have improved our understanding of the redox proteome, the widespread adoption of these methods is often hindered by complex sample preparation workflows, prohibitive cost of isotopic labeling reagents, and requirements for custom data analysis workflows. Here, we present the SP3-Rox redox proteomics method that combines tailored low cost isotopically labeled capture reagents with SP3 sample cleanup to achieve high throughput and high coverage proteome-wide identification of redox-sensitive cysteines. By implementing a customized workflow in the free FragPipe computational pipeline, we achieve accurate MS1-based quantitation, including for peptides containing multiple cysteine residues. Application of the SP3-Rox method to cellular proteomes identified cysteines sensitive to the oxidative stressor GSNO and cysteine oxidation state changes that occur during T cell activation.