Redox sensitive epidithiodioxopiperazines in biological mechanisms of toxicity

Abstract

Epidithiodioxopiperazines (ETPs) are a class of secondary metabolites characterized by a bridged disulfide linkage across the α,α′-positions of the dioxopiperazine ring. This class of compounds displays a range of biological activities, attributed to the sulfur moiety in the oxidized disulfide form and/or the reduced dithiol form. The underlying mechanisms of toxicity of the ETP metabolites are still a matter of debate and this review presents an overview of the evidence for the possible pathways of toxicity.     

Oxidative Folding of Conopeptides Modified by <Emphasis Type="Italic">Conus</Emphasis> Protein Disulfide Isomerase

Protein disulfide isomerase is a type of enzyme that catalyses the oxidation, isomerization and reduction of disulfide bonds. Conotoxins that containing disulfide bonds are likely substrates of protein disulfide isomerise. Here, we cloned 12 protein disulfide isomerise genes from 12 different cone snail species that inhabited the sea near Sanya in China. The full-length amino acid sequences of these protein disulfide isomerase genes share a high degree of homology, including the same -CGHC- active site sequence and -RDEL- endoplasmic reticulum retention signal. To obtain enough conus protein disulfide isomerase for functional studies, we constructed the expression vector pET28a-sPDI. Conus protein disulfide isomerase was successfully expressed using Escherichia coli expression system and purified using chromatography method of affinity chromatography. The recombinant conus protein disulfide isomerase showed the ability to catalyse disulfide bond formation and rearrangement in the lysozyme enzyme activity assay. The role of conus protein disulfide isomerase in the in vitro oxidative folding of conotoxins was investigated using synthetic linear conotoxin lt14a, a peptide composed of 13 amino acids. It was confirmed by high performance liquid chromatography and mass spectrometry analysis that conus protein disulfide isomerase can catalyse the disulfide bond formation of linear lt14a. Then, conus protein disulfide isomerase was acted as a fusion partner during the production of engineered peptidyl-prolyl cis–trans isomerase and lt14a derived from cone snails. It was shown that peptidyl-prolyl cis–trans isomerase and conotoxin lt14a are successfully expressed in a highly soluble form by fusion with conus protein disulfide isomerase. Thus, conus protein disulfide isomerase functions not only as an enzyme that catalyses oxidative process but also a fusion partner in recombinant conotoxin expression.     

In vitro bone marrow granulocyte-macrophage progenitor cultures in the assessment of hematotoxic potential of the new drugs

In pharmaceutical research, in vitro toxicity tests, for assessing the potential toxicity of new chemical entities are necessary in the early stages of the developmental process, when no information is available about the metabolism or even the target organ toxicity of the compounds to be tested. In vitro specific organ toxicity tests, such as the granulocyte-macrophage colony-forming unit (CFU-GM) clonogenic assay, are useful tools for predicting the adverse effects of new compounds on the blood-forming system, provided that some reference points are available, e.g., toxicological information about compounds belonging to the same chemical class and structure-activity relationship data. Furthermore, when no information is available about metabolism, the in vitro system should cover as many possibilities as possible, to avoid false positive or false negative results. In fact, while many compounds are metabolized to a variety of inactive chemical species, some undergo bioactivation to form more active metabolites. The addition of a metabolic activation system to the CFU-GM assay enables assessment of direct and metabolism-mediated toxicity. The regulatory agencies and industry value the concept of assays performed with and without metabolic activation, since they often have to take decisions about compounds with unknown mechanisms of action. CFU-GM assay, designed in this way, is an example of such a mechanism-naive assay. It has been suggested that, for new compounds, metabolites should be generated and tested both in the presence and in the absence of the parent compound itself, to identify the possible contribution of metabolites to the hematotoxicity observed, and to determine whether there is any synergistic or antagonistic effect between metabolites and the parent compound that might affect hematotoxicity in vivo. Various approaches can be used to obtain such information.     

Structural Alerts,Reactive Metabolites,and Protein Covalent Binding: How Reliable Are These Attributes as Predictors of Drug Toxicity?

In an increasing number of cases, a deeper understanding of the biochemical basis for idiosyncratic adverse drug reactions (IADRs) has aided to replace a vague perception of a chemical class effect with a sharper picture of individual molecular peculiarity. Considering that IADRs are too complex to duplicate in a test tube, and their idiosyncratic nature precludes prospective clinical studies, it is currently impossible to predict which new drugs will be associated with a significant incidence of toxicity. Because it is now widely appreciated that reactive metabolites, as opposed to the parent molecules from which they are derived, are responsible for the pathogenesis of some IADRs, the propensity of drug candidates to form reactive metabolites is generally considered a liability. Procedures have been implemented to monitor reactive‐metabolite formation in discovery with the ultimate goal of eliminating or minimizing the liability via rational structural modification of the problematic chemical series. While such mechanistic studies have provided retrospective insight into the metabolic pathways which lead to reactive metabolite formation with toxic compounds, their ability to accurately predict the IADR potential of new drug candidates has been challenged. There are several instances of drugs that form reactive metabolites, but only a fraction thereof cause toxicity. This review article will outline current approaches to evaluate bioactivation potential of new compounds with particular emphasis on the advantages and limitation of these assays. Plausible reason(s) for the excellent safety record of certain drugs susceptible to bioactivation will also be explored and should provide valuable guidance in the use of reactive‐metabolite assessments when nominating drug candidates for development.     

Amyotrophic lateral sclerosis mutations have the greatest destabilizing effect on the apo- and reduced form of SOD1, leading to unfolding and oxidative aggregation

Mutant forms of Cu,Zn-superoxide dismutase (SOD1) that cause familial amyotrophic lateral sclerosis (ALS) exhibit toxicity that promotes the death of motor neurons. Proposals for the toxic properties typically involve aberrant catalytic activities or protein aggregation. The striking thermodynamic stability of mature forms of the ALS mutant SOD1 (Tm>70 degrees C) is not typical of protein aggregation models that involve unfolding. Over 44 states of the polypeptide are possible, depending upon metal occupancy, disulfide status, and oligomeric state; however, it is not clear which forms might be responsible for toxicity. Recently the intramolecular disulfide has been shown to be required for SOD1 activity, leading us to examine these states of several disease-causing SOD1 mutants. We find that ALS mutations have the greatest effect on the most immature form of SOD1, destabilizing the metal-free and disulfide-reduced polypeptide to the point that it is unfolded at physiological temperatures (Tm<37 degrees C). We also find that immature states of ALS mutant (but not wild type) proteins readily form oligomers at physiological concentrations. Furthermore, these oligomers are more susceptible to mild oxidative stress, which promotes incorrect disulfide cross-links between conserved cysteines and drives aggregation. Thus it is the earliest disulfide-reduced polypeptides in the SOD1 assembly pathway that are most destabilized with respect to unfolding and oxidative aggregation by ALS-causing mutations.     

Folding studies of Cox17 reveal an important interplay of cysteine oxidation and copper binding

Cox17 is a key mitochondrial copper chaperone involved in the assembly of cytochrome c oxidase (COX). The NMR solution structure of the oxidized apoCox17 isoform consists of a coiled-coil conformation stabilized by two disulfide bonds involving Cys(26)/Cys(57) and Cys(36)/Cys(47). This appears to be a conserved tertiary fold of a class of proteins, localized within the mitochondrial intermembrane space, that contain a twin Cys-x(9)-Cys sequence motif. An isomerization of one disulfide bond from Cys(26)/Cys(57) to Cys(24)/Cys(57) is required prior to Cu(I) binding to form the Cu(1)Cox17 complex. Upon further oxidation of the apo-protein, a form with three disulfide bonds is obtained. The reduction of all disulfide bonds provides a molten globule form that can convert to an additional conformer capable of binding up to four Cu(I) ions in a polycopper cluster. This form of the protein is oligomeric. These properties are framed within a complete model of mitochondrial import and COX assembly.     

Gamma-interferon-inducible lysosomal thiol reductase (GILT). Maturation, activity, and mechanism of action

We recently identified a gamma-interferon-inducible lysosomal thiol reductase (GILT), constitutively expressed in antigen-presenting cells, that catalyzes disulfide bond reduction both in vitro and in vivo and is optimally active at acidic pH. GILT is synthesized as a 35-kDa precursor, and following delivery to major histocompatibility complex (MHC) class II-containing compartments (MIICs), is processed to the mature 30-kDa form via cleavage of N- and C-terminal propeptides. The generation of MHC class II epitopes requires both protein denaturation and reduction of intra- and inter-chain disulfide bonds prior to proteolysis. GILT may be important in disulfide bond reduction of proteins delivered to MIICs and consequently in antigen processing. In this report we show that, like its mature form, precursor GILT reduces disulfide bonds with an acidic pH optimum, suggesting that it may also be involved in disulfide bond reduction in the endocytic pathway. We also show that processing of precursor GILT can be mediated by multiple lysosomal proteases and provide evidence that the mechanism of action of GILT resembles that of other thiol oxidoreductases.     

Mitochondrial Ccs1 contains a structural disulfide bond crucial for the import of this unconventional substrate by the disulfide relay system

The copper chaperone for superoxide dismutase 1 (Ccs1) provides an important cellular function against oxidative stress. Ccs1 is present in the cytosol and in the intermembrane space (IMS) of mitochondria. Its import into the IMS depends on the Mia40/Erv1 disulfide relay system, although Ccs1 is, in contrast to typical substrates, a multidomain protein and lacks twin Cx(n)C motifs. We report on the molecular mechanism of the mitochondrial import of Saccharomyces cerevisiae Ccs1 as the first member of a novel class of unconventional substrates of the disulfide relay system. We show that the mitochondrial form of Ccs1 contains a stable disulfide bond between cysteine residues C27 and C64. In the absence of these cysteines, the levels of Ccs1 and Sod1 in mitochondria are strongly reduced. Furthermore, C64 of Ccs1 is required for formation of a Ccs1 disulfide intermediate with Mia40. We conclude that the Mia40/Erv1 disulfide relay system introduces a structural disulfide bond in Ccs1 between the cysteine residues C27 and C64, thereby promoting mitochondrial import of this unconventional substrate. Thus the disulfide relay system is able to form, in addition to double disulfide bonds in twin Cx(n)C motifs, single structural disulfide bonds in complex protein domains.     

The Familial British Dementia Mutation Promotes Formation of Neurotoxic Cystine Cross-linked Amyloid Bri (ABri) Oligomers

Familial British dementia (FBD) is an inherited neurodegenerative disease believed to result from a mutation in the BRI2 gene. Post-translational processing of wild type BRI2 and FBD-BRI2 result in the production of a 23-residue long Bri peptide and a 34-amino acid long ABri peptide, respectively, and ABri is found deposited in the brains of individuals with FBD. Similarities in the neuropathology and clinical presentation shared by FBD and Alzheimer disease (AD) have led some to suggest that ABri and the AD-associated amyloid β-protein (Aβ) are molecular equivalents that trigger analogous pathogenic cascades. But the sequences and innate properties of ABri and Aβ are quite different, notably ABri contains two cysteine residues that can form disulfide bonds. Thus we sought to determine whether ABri was neurotoxic and if this activity was regulated by oxidation and/or aggregation. Crucially, the type of oxidative cross-linking dramatically influenced both ABri aggregation and toxicity. Cyclization of Bri and ABri resulted in production of biologically inert monomers that showed no propensity to assemble, whereas reduced ABri and reduced Bri aggregated forming thioflavin T-positive amyloid fibrils that lacked significant toxic activity. ABri was more prone to form inter-molecular disulfide bonds than Bri and the formation of covalently stabilized ABri oligomers was associated with toxicity. These results suggest that extension of the C-terminal of Bri causes a shift in the type of disulfide bonds formed and that structures built from covalently cross-linked oligomers can interact with neurons and compromise their function and viability.     

Protection of arsenic-induced testicular oxidative stress by arjunolic acid

Arsenic-induced tissue damage is a major concern to the human population. An impaired antioxidant defense mechanism followed by oxidative stress is the major cause of arsenic-induced toxicity, which can lead to reproductive failure. The present study was carried out to investigate the preventive role of arjunolic acid, a triterpenoid saponin isolated from the bark of Terminalia arjuna, against arsenic-induced testicular damage in mice. Administration of arsenic (in the form of sodium arsenite, NaAsO(2), at a dose of 10 mg/kg body weight) for 2 days significantly decreased the intracellular antioxidant power, the activities of the antioxidant enzymes, as well as the levels of cellular metabolites. In addition, arsenic intoxication enhanced testicular arsenic content, lipid peroxidation, protein carbonylation and the level of glutathione disulfide (GSSG). Exposure to arsenic also caused significant degeneration of the seminiferous tubules with necrosis and defoliation of spermatocytes. Pretreatment with arjunolic acid at a dose of 20 mg/kg body weight for 4 days could prevent the arsenic-induced testicular oxidative stress and injury to the histological structures of the testes. Arjunolic acid had free radical scavenging activity in a cell-free system and antioxidant power in vivo. In summary, the results suggest that the chemopreventive role of arjunolic acid against arsenic-induced testicular toxicity may be due to its intrinsic antioxidant property.     

Characterization and analysis of scFv-IgG bispecific antibody size variants

ABSTRACT

Bispecific antibodies are an emergent class of biologics that is of increasing interest for therapeutic applications. In one bispecific antibody format, single-chain variable fragments (scFv) are linked to or inserted in different locations of an intact immunoglobulin G (IgG) molecule to confer dual epitope binding. To improve biochemical stability, cysteine residues are often engineered on the heavy- and light-chain regions of the scFv to form an intrachain disulfide bond. Although this disulfide bond often improves stability, it can also introduce unexpected challenges to manufacturing or development. We report size variants that were observed for an appended scFv-IgG bispecific antibody. Structural characterization studies showed that the size variants resulted from the engineered disulfide bond on the scFv, whereby the engineered disulfide was found to be either open or unable to form an intrachain disulfide bond due to cysteinylation or glutathionylation of the cysteines. Furthermore, the scFv engineered cysteines also formed intermolecular disulfide bonds, leading to the formation of highly stable dimers and aggregates. Because both the monomer variants and dimers showed lower bioactivity, they were considered to be product-related impurities that must be monitored and controlled. To this end, we developed and optimized a robust, precise, and accurate high-resolution size-exclusion chromatographic method, using a statistical design-of-experiments methodology.     

Conformational landscape and pathway of disulfide bond reduction of human alpha defensin

Human alpha defensins are a class of antimicrobial peptides with additional antiviral activity. Such antimicrobial peptides constitute a major part of mammalian innate immunity. Alpha defensins contain six cysteines, which form three well defined disulfide bridges under oxidizing conditions. Residues C3-C31, C5-C20, and C10-C30 form disulfide pairs in the native structure of the peptide. The major tissue in which HD5 is expressed is the crypt of the small intestine, an anaerobic niche that should allow for substantial pools of both oxidized and (partly) reduced HD5. We used ion mobility coupled to mass spectrometry to track the structural changes in HD5 upon disulfide bond reduction. We found evidence of stepwise unfolding of HD5 with sequential reduction of the three disulfide bonds. Alkylation of free cysteines followed by tandem mass spectrometry of the corresponding partially reduced states revealed a dominant pathway of reductive unfolding. The majority of HD5 unfolds by initial reduction of C5-C20, followed by C10-C30 and C3-C31. We find additional evidence for a minor pathway that starts with reduction of C3-C31, followed by C5-C20 and C10-C30. Our results provide insight into the pathway and conformational landscape of disulfide bond reduction in HD5.     

Oxidation of thiamine on reaction with nitrogen dioxide generated by ferric myoglobin and hemoglobin in the presence of nitrite and hydrogen peroxide

It is shown that nitrogen dioxide oxidizes thiamine to thiamine disulfide, thiochrome, and oxodihydrothiochrome (ODTch). The latter is formed during oxidation of thiochrome by nitrogen dioxide. Nitrogen dioxide was produced by incubation of nitrite with horse ferric myoglobin and human hemoglobin in the presence of hydrogen peroxide. After addition of tyrosine or phenol to aqueous solutions containing oxoferryl forms of the hemoproteins, thiamine, and nitrite, the yield of thiochrome greatly increased, whereas the yield of ODTch decreased. In the presence of high concentrations of tyrosine or phenol compounds ODTch was not formed at all. The neutral form of thiamine with the closed thiazole cycle and minor tricyclic form of thiamine do not enter the heme pocket of the protein and do not interact with the oxoferryl heme complex Fe(IV=O) or porphyrin radical. The tricyclic form of thiamine is oxidized to thiochrome by tyrosyl radicals located on the surface of the hemoprotein. The thiol form of thiamine is oxidized to thiamine disulfide by both hemoprotein tyrosyl radicals and oxoferryl heme complexes. Nitrite and also tyrosine, tyramine, and phenol readily penetrate into the heme pocket of the protein and reduce the oxyferryl complex to ferric cation. These reactions yield nitrogen dioxide as well as tyrosyl and phenoxyl radicals of tyrosine molecules and phenol compounds, respectively. Tyrosyl and phenoxyl radicals of low molecular weight compounds oxidize thiamine only to thiochrome and thiamine disulfide. The effect of oxoferryl forms of myoglobin and hemoglobin, nitrogen dioxide, and phenol on thiamine oxidative transformation as well as antioxidant properties of the hydrophobic thiamine metabolites thiochrome and ODTch are discussed.     

Bioactivation of xenobiotics by formation of toxic glutathione conjugates

Evidence has been accumulating that several classes of compounds are converted by glutathione conjugate formation to toxic metabolites. The aim of this review is to summarize the current knowledge on the biosynthesis and toxicity of glutathione S-conjugates derived from halogenated alkanes, halogenated alkenes, and hydroquinones and quinones. Different types of toxic glutathione conjugates have been identified and will be discussed in detail: (i) conjugates which are transformed to electrophilic sulfur mustards, (ii) conjugates which are converted to toxic metabolites in an enzyme-catalyzed multistep mechanism, (iii) conjugates which serve as a transport form for toxic quinones and (iv) reversible glutathione conjugate formation and release of the toxic agent in cell types with lower glutathione concentrations. The kidney is the main, with some compounds the exclusive, target organ for compounds metabolized by pathways (i) to (iii). Selective toxicity to the kidney is easily explained due to the capability of the kidney to accumulate intermediates formed by processing of S-conjugates and to bioactivate these intermediates to toxic metabolites. The influences of other factors participating in the renal susceptibility are discussed.     

… -ome sweet -ome

Abstract

Arsenic-induced tissue damage is a major concern to the human population. An impaired antioxidant defense mechanism followed by oxidative stress is the major cause of arsenic-induced toxicity, which can lead to reproductive failure. The present study was carried out to investigate the preventive role of arjunolic acid, a triterpenoid saponin isolated from the bark of Terminalia arjuna, against arsenic-induced testicular damage in mice. Administration of arsenic (in the form of sodium arsenite, NaAsO₂, at a dose of 10 mg/kg body weight) for 2 days significantly decreased the intracellular antioxidant power, the activities of the antioxidant enzymes, as well as the levels of cellular metabolites. In addition, arsenic intoxication enhanced testicular arsenic content, lipid peroxidation, protein carbonylation and the level of glutathione disulfide (GSSG). Exposure to arsenic also caused significant degeneration of the seminiferous tubules with necrosis and defoliation of spermatocytes. Pretreatment with arjunolic acid at a dose of 20 mg/kg body weight for 4 days could prevent the arsenic-induced testicular oxidative stress and injury to the histological structures of the testes. Arjunolic acid had free radical scavenging activity in a cell-free system and antioxidant power in vivo. In summary, the results suggest that the chemopreventive role of arjunolic acid against arsenic-induced testicular toxicity may be due to its intrinsic antioxidant property.     

Modulation of Protein Fermentation Does Not Affect Fecal Water Toxicity: A Randomized Cross-Over Study in Healthy Subjects

Objective

Protein fermentation results in production of metabolites such as ammonia, amines and indolic, phenolic and sulfur-containing compounds. In vitro studies suggest that these metabolites might be toxic. However, human and animal studies do not consistently support these findings. We modified protein fermentation in healthy subjects to assess the effects on colonic metabolism and parameters of gut health, and to identify metabolites associated with toxicity.

Design

After a 2-week run-in period with normal protein intake (NP), 20 healthy subjects followed an isocaloric high protein (HP) and low protein (LP) diet for 2 weeks in a cross-over design. Protein fermentation was estimated from urinary p-cresol excretion. Fecal metabolite profiles were analyzed using GC-MS and compared using cluster analysis. DGGE was used to analyze microbiota composition. Fecal water genotoxicity and cytotoxicity were determined using the Comet assay and the WST-1-assay, respectively, and were related to the metabolite profiles.

Results

Dietary protein intake was significantly higher during the HP diet compared to the NP and LP diet. Urinary p-cresol excretion correlated positively with protein intake. Fecal water cytotoxicity correlated negatively with protein fermentation, while fecal water genotoxicity was not correlated with protein fermentation. Heptanal, 3-methyl-2-butanone, dimethyl disulfide and 2-propenyl ester of acetic acid are associated with genotoxicity and indole, 1-octanol, heptanal, 2,4-dithiapentane, allyl-isothiocyanate, 1-methyl-4-(1-methylethenyl)-benzene, propionic acid, octanoic acid, nonanoic acid and decanoic acid with cytotoxicity.

Conclusion

This study does not support a role of protein fermentation in gut toxicity. The identified metabolites can provide new insight into colonic health.

Trial Registration

ClinicalTrial.gov {"type":"clinical-trial","attrs":{"text":"NCT01280513","term_id":"NCT01280513"}}NCT01280513     

Disulfide Bond Formation in the Mammalian Endoplasmic Reticulum

The formation of disulfide bonds between cysteine residues occurs during the folding of many proteins that enter the secretory pathway. As the polypeptide chain collapses, cysteines brought into proximity can form covalent linkages during a process catalyzed by members of the protein disulfide isomerase family. There are multiple pathways in mammalian cells to ensure disulfides are introduced into proteins. Common requirements for this process include a disulfide exchange protein and a protein oxidase capable of forming disulfides de novo. In addition, any incorrect disulfides formed during the normal folding pathway are removed in a process involving disulfide exchange. The pathway for the reduction of disulfides remains poorly characterized. This work will cover the current knowledge in the field and discuss areas for future investigation.One of the characteristics of proteins that enter the secretory pathway is that they frequently contain covalent linkages called disulfide bonds within and between constituent polypeptide chains. The presence of these linkages is thought to confer stability when secreted proteins are exposed to the extracellular milieu or when membrane proteins are recycled through acidic endocytic compartments. In addition to structural disulfides it is now clear that a number of proteins use the formation and breaking of disulfides as a mechanism for regulation of activity (Schwertassek et al. 2007). Hence, it is important that we have a clear understanding of how correct disulfides are formed within proteins both during the protein folding process and to regulate protein function. The focus of this article will be on how correct disulfides are introduced into proteins within the secretory pathway, specifically within the endoplasmic reticulum (ER) during folding and assembly.The formation of disulfides within polypeptides begins as the protein is being cotranslationally translocated into the ER (Chen et al. 1995). The initial collapse of the polypeptide and formation of secondary structure brings cysteine residues into close enough proximity for them to form disulfides. Correct disulfide formation requires enzymes to both introduce disulfides between proximal cysteines and to reduce disulfides that form during folding but that are not present in the final native structure (Jansens et al. 2002). In addition, proteins that do not fold correctly are targeted for degradation and may require their disulfides to be broken before dislocation across the ER membrane into the cytosol (Ushioda et al. 2008). Hence, there must be a reduction and oxidation pathway present in the ER to ensure that native disulfides form and nonnative disulfides are broken during protein folding.Central to both reduction and oxidation pathways is the protein disulfide isomerase (PDI) family of enzymes (Ellgaard and Ruddock 2005) that are capable of exchanging disulfides with their substrate proteins (Fig. 1). Whether disulfide exchange results in the formation or breaking of a disulfide depends on the relative stability of the disulfides in the enzyme and substrate. To drive the formation of disulfides, the PDI family member must itself be oxidized. It is now clear that there are a number of ways for the disulfide exchange proteins to be oxidized by specific oxidases. Importantly, these oxidases do not introduce disulfides into nascent polypeptide chains; rather, they specifically oxidize members of the PDI family. The exception to this rule is the enzyme quiescin sulfydryl oxidase (QSOX; see below). The pathway for disulfide reduction is not as well characterized. It is known that the PDI family members can be reduced by the low molecular mass thiol glutathione (GSH) (Chakravarthi and Bulleid 2004; Jessop and Bulleid 2004; Molteni et al. 2004) but no enzymatic process for reduction has been identified. The glutathione redox balance within the ER is significantly more oxidized than in the cytosol (Hwang et al. 1992; Dixon et al. 2008), indicating that GSH is actively oxidized to glutathione disulfide either during the reduction of PDI family members or by reducing disulfides in nascent polypeptides directly. However, there is currently no clear indication as to how glutathione disulfide is itself reduced.Open in a separate windowFigure 1.PDI family of enzymes catalyzes disulfide exchange reactions in the endoplasmic reticulum. Nascent polypeptide chains are cotranslationally translocated across the ER membrane whereupon cysteines in close proximity can form disulfides. The reaction is catalyzed by members of the PDI family (depicted as PDI) by a disulfide exchange reaction resulting in the reduction of the PDI active site. If nonnative disulfides are formed these can be reduced by the reverse disulfide exchange reaction, resulting in the oxidation of the PDI active site.Both the formation and breaking of disulfides can be thought of as electron transport pathways that require suitable electron acceptors or donors to drive the flow of electrons. For the purposes of this article the two pathways will be discussed separately, but it should be appreciated that each pathway occurs within the same organelle so the possibility of crossover between them is real. Whether futile redox reactions occur between the pathways is unclear but any kinetic segregation of the pathways will be highlighted where it is known to occur.     

Disulfide recognition in an optimized threading potential

An energy potential is constructed and trained to succeed in fold recognition for the general population of proteins as well as an important class which has previously been problematic: small, disulfide-bearing proteins. The potential is modeled on solvation, with the energy a function of side chain burial and the number of disulfide bonds. An accurate disulfide recognition algorithm identifies cysteine pairs which have the appropriate orientation to form a disulfide bridge. The potential has 22 energy parameters which are optimized so the Protein Data Bank (PDB) structure for each sequence in a training set is the lowest in energy out of thousands of alternative structures. One parameter per amino acid type reflects burial preference and a single parameter is used in an overpacking term. Additionally, one optimized parameter provides a favorable contribution for each disulfide identified in a given protein structure. With little training, the potential is >80% accurate in ungapped threading tests using a variety of proteins. The same level of accuracy is observed in a threading test of small proteins which have disulfide bonds. Importantly, the energy potential is also successful with proteins having uncrosslinked cysteines.     

Acetaminophen toxicity in lymphocytes heterozygous for glutathione synthetase deficiency

We have studied the effects of acetaminophen metabolites generated by a murine hepatic microsomal system on lymphocytes from two subjects heterozygous for glutathione synthetase deficiency. Heterozygous cells exhibited greater dose-related toxicity than controls. Following a 2-h incubation with acetaminophen and the microsomal system, cells were washed and incubated for 16 h in the presence or absence of N-acetylcysteine, the standard antidote for acetaminophen toxicity. In control cells, glutathione content was replenished to nearly base-line values and toxicity was prevented. N-Acetylcysteine thus prevented toxicity even after covalent binding of acetaminophen metabolites had occurred. Heterozygous cells failed to use N-acetylcysteine as efficiently to resynthesize glutathione, and the cells were not protected from acetaminophen toxicity. Heterozygotes may be at increased risk of toxicity from drugs whose metabolites are detoxified by glutathione conjugation.     

Predicting disulfide connectivity patterns

Disulfide bonds play an important role in stabilizing protein structure and regulating protein function. Therefore, the ability to infer disulfide connectivity from protein sequences will be valuable in structural modeling and functional analysis. However, to predict disulfide connectivity directly from sequences presents a challenge to computational biologists due to the nonlocal nature of disulfide bonds, i.e., the close spatial proximity of the cysteine pair that forms the disulfide bond does not necessarily imply the short sequence separation of the cysteine residues. Recently, Chen and Hwang (Proteins 2005;61:507-512) treated this problem as a multiple class classification by defining each distinct disulfide pattern as a class. They used multiple support vector machines based on a variety of sequence features to predict the disulfide patterns. Their results compare favorably with those in the literature for a benchmark dataset sharing less than 30% sequence identity. However, since the number of disulfide patterns grows rapidly when the number of disulfide bonds increases, their method performs unsatisfactorily for the cases of large number of disulfide bonds. In this work, we propose a novel method to represent disulfide connectivity in terms of cysteine pairs, instead of disulfide patterns. Since the number of bonding states of the cysteine pairs is independent of that of disulfide bonds, the problem of class explosion is avoided. The bonding states of the cysteine pairs are predicted using the support vector machines together with the genetic algorithm optimization for feature selection. The complete disulfide patterns are then determined from the connectivity matrices that are constructed from the predicted bonding states of the cysteine pairs. Our approach outperforms the current approaches in the literature.