Stable Disulfide Bonds Research Articles

Low-Molecular-Weight Oxidants Involved in Disulfide Bond Formation

The biogenesis of most secreted and outer membrane proteins involves the formation of structure stabilizing disulfide bonds. Hence knowledge of the mechanisms for their formation is critical for understanding a myriad of cellular processes and associated disease states. Until recently it was thought that members of the Ero1 sulfhydryl oxidase family were responsible for catalyzing the majority of disulfide bond formation in the endoplasmic reticulum. However, multiple eukaryotic organisms are now known to show no or minor phenotypes when these enzymatic pathways are disrupted, suggesting that other pathways can catalyze disulfide bond formation to an extent sufficient to maintain normal physiology. This lack of a strong phenotype raises multiple questions regarding what pathways are acting and whether they themselves constitute the major route for disulfide bond formation. This review critically examines the potential low molecular oxidants that maybe involved in the catalyzed or noncatalyzed formation of disulfide bonds, with an emphasis on the mammalian endoplasmic reticulum, via an examination of their thermodynamics, kinetics, and availability and gives pointers to help guide future experimental work.

Synthesis and Biological Evaluation of Antibody Conjugates of Phosphate Prodrugs of Cytotoxic DNA Alkylators for the Targeted Treatment of Cancer

The synthesis and biological evaluation of phosphate prodrugs of analogues of 1 (CC-1065) and their conjugates with antibodies are described. The phosphate group on the 1,2,9,9a-tetrahydrocyclopropa[c]benz[e]indol-4-one (CBI) portion of the compounds confers enhanced solubility and stability in aqueous solutions. In the presence of phosphatases, these compounds convert into active DNA-alkylating agents. The synthesis of the prodrugs was achieved sequentially through coupling of CBI with a bis-indolyl moiety, followed by attachment of a thiol-containing linker, and conversion of the hydroxyl group of CBI into a phosphate prodrug. The linkers incorporated into the prodrugs enable conjugation to an antibody via either a stable disulfide or thioether bond, in aqueous buffer solutions containing as little as 5% organic cosolvent, resulting in exclusively monomeric and stable antibody-cytotoxic prodrug conjugates. Two disulfide-containing linkers differing in the degree of steric hindrance were used in antibody conjugates to test the effect of different rates of intracellular disulfide cleavage and effector release on biological activity. The prodrugs can be converted to the active cytotoxic compounds through the action of endogenous phosphatases. Antibody-prodrug conjugates displayed potent antigen-selective cytotoxic activity in vitro and antitumor activity in vivo.

Structure Based Protein Models and In Vitro Experiments to Investigate the Folding Landscape of the Human Obesity Protein Leptin

Jeffery Friedman discovered the human obesity gen in 1994. The expressed hormone leptin plays a key role in regulating energy intake/expenditure through the JAK/STAT signaling pathway. Even though, biologically, it is a well-studied protein not much is known about the biophysical properties.The mature protein is 146 residues (16kDa) buildup of 5 α-helixes and one stabilizing disulfide bond. This disulfide bridge encloses the C-terminal cysteine with residue 96 into a 50 residues long loop, creating a ‘so-called’ cysteine knot.In this research we explore the free energy landscape through structure-based protein models (Cα- and all-atom) and experiments (CD and fluorescence). The result shows that leptin can fold both in its reduced- and oxidized state. Even though the oxidized protein has a non-trivial pathway, where the N-terminal has to ‘thread’ through the loop to reach the native state, the folding landscape looks almost the same as for the reduced protein. The first event of folding is to form the loop or horseshoe (in the reduced state) constructed of α4 and α5 to stabilize the structure. Secondly, α3 is pulled into position: Either by a slipknot, backing into the loop in the oxidized state or by being pulled back to the correct conformation behind the horseshoe in the reduced state. The dragging force for this slipknot event is pulled from the energetic gain of zipping α2 together with α5. The last step is to fold α1 into its native conformation in the back of the structure between α5 and α2.

Hepatitis C Virus (HCV) Envelope Glycoproteins E1 and E2 Contain Reduced Cysteine Residues Essential for Virus Entry

The HCV envelope glycoproteins E1 and E2 contain eight and 18 highly conserved cysteine residues, respectively. Here, we examined the oxidation state of E1E2 heterodimers incorporated into retroviral pseudotyped particles (HCVpp) and investigated the significance of free sulfhydryl groups in cell culture-derived HCV (HCVcc) and HCVpp entry. Alkylation of free sulfhydryl groups on HCVcc/pp with a membrane-impermeable sulfhydryl-alkylating reagent 4-(N-maleimido)benzyl-α-trimethylammonium iodide (M135) prior to virus attachment to cells abolished infectivity in a dose-dependent manner. Labeling of HCVpp envelope proteins with EZ-Link maleimide-PEG2-biotin (maleimide-biotin) detected free thiol groups in both E1 and E2. Unlike retroviruses that employ disulfide reduction to facilitate virus entry, the infectivity of alkylated HCVcc could not be rescued by addition of exogenous reducing agents. Furthermore, the infectivity of HCVcc bound to target cells was not affected by addition of M135 indicative of a change in glycoprotein oxidation state from reduced to oxidized following virus attachment to cells. By contrast, HCVpp entry was reduced by 61% when treated with M135 immediately following attachment to cells, suggesting that the two model systems might demonstrate variations in oxidation kinetics. Glycoprotein oxidation was not altered following binding of HCVpp incorporated E1E2 to soluble heparin or recombinant CD81. These results suggest that HCV entry is dependent on the presence of free thiol groups in E1 and E2 prior to cellular attachment and reveals a new essential component of the HCV entry process.

Oxidative folding: recent developments.

Disulfide bond formation in proteins is an effective tool of both structure stabilization and redox regulation. The prokaryotic periplasm and the endoplasmic reticulum of eukaryotes were long considered as the only compartments for enzyme mediated formation of stable disulfide bonds. Recently, the mitochondrial intermembrane space has emerged as the third protein-oxidizing compartment. The classic view on the mechanism of oxidative folding in the endoplasmic reticulum has also been reshaped by new observations. Moreover, besides the structure stabilizing function, reversible disulfide bridge formation in some proteins of the endoplasmic reticulum, seems to play a regulatory role. This review briefly summarizes the present knowledge of the redox systems supporting oxidative folding, emphasizing recent developments.

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.

Disulfide-Linked Antibody−Maytansinoid Conjugates: Optimization of In Vivo Activity by Varying the Steric Hindrance at Carbon Atoms Adjacent to the Disulfide Linkage

In this report, we describe the synthesis of a panel of disulfide-linked huC242 (anti-CanAg) antibody maytansinoid conjugates (AMCs), which have varying levels of steric hindrance around the disulfide bond, in order to investigate the relationship between stability to reduction of the disulfide linker and antitumor activity of the conjugate in vivo. The conjugates were first tested for stability to reduction by dithiothreitol in vitro and for plasma stability in CD1 mice. It was found that the conjugates having the more sterically hindered disulfide linkages were more stable to reductive cleavage of the maytansinoid in both settings. When the panel of conjugates was tested for in vivo efficacy in two human colon cancer xenograft models in SCID mice, it was found that the conjugate with intermediate disulfide bond stability having two methyl groups on the maytansinoid side of the disulfide bond and no methyl groups on the linker side of the disulfide bond (huC242-SPDB-DM4) displayed the best efficacy. The ranking of in vivo efficacies of the conjugates was not predicted by their in vitro potencies, since all conjugates were highly active in vitro, including a huC242-SMCC-DM1 conjugate with a noncleavable linkage which showed only marginal activity in vivo. These data suggest that factors in addition to intrinsic conjugate potency and conjugate half-life in plasma influence the magnitude of antitumor activity observed for an AMC in vivo. We provide evidence that bystander killing of neighboring nontargeted tumor cells by diffusible cytotoxic metabolites produced from target cell processing of disulfide-linked antibody-maytansinoid conjugates may be one additional factor contributing to the activity of these conjugates in vivo.

Cytosolic Disulfide Bond Formation in Cells Infected with Large Nucleocytoplasmic DNA Viruses

Proteins that have evolved to contain stabilizing disulfide bonds generally fold in a membrane-delimited compartment in the cell [i.e., the endoplasmic reticulum (ER) or the mitochondrial intermembrane space (IMS)]. These compartments contain sulfhydryl oxidase enzymes that catalyze the pairing and oxidation of cysteine residues. In contrast, most proteins in a healthy cytosol are maintained in reduced form through surveillance by NADPH-dependent reductases and the lack of sulfhydryl oxidases. Nevertheless, one of the core functionalities that unify the broad and diverse set of nucleocytoplasmic large DNA viruses (NCLDVs) is the ability to catalyze disulfide formation in the cytosol. The substrates of this activity are proteins that contribute to the assembly, structure, and infectivity of the virions. If the last common ancestor of NCLDVs was present during eukaryogenesis as has been proposed, it is interesting to speculate that viral disulfide bond formation pathways may have predated oxidative protein folding in intracellular organelles.

Large-scale production of a disulfide-stabilized constitutively active mutant opsin

Previous studies of constitutively activated mutants of opsin in the absence of chromophore were carried out in crude cell membranes because such mutants could not be recovered in a detergent-solubilized form in the active state. We employed a strategy in which a stabilizing disulfide bond allowed for successful purification of a constitutively activated mutant opsin, N2C/E113Q/M257Y/D282C, solubilized in nonionic detergent from mammalian cell culture. The purified mutant opsin is able to activate transducin to a higher degree than opsin and may prove useful for future structural studies of the active state of GPCRs.

Disulfide Bond Stabilization of the Hexameric Capsomer of Human Immunodeficiency Virus

The human immunodeficiency virus type 1 capsid is modeled as a fullerene cone that is composed of ∼250 hexamers and 12 pentamers of the viral CA protein. Structures of CA hexamers have been difficult to obtain because the hexamer-stabilizing interactions are inherently weak, and CA tends to spontaneously assemble into capsid-like particles. Here, we describe a two-step biochemical strategy to obtain soluble CA hexamers for crystallization. First, the hexamer was stabilized by engineering disulfide cross-links (either A14C/E45C or A42C/T54C) between the N-terminal domains of adjacent subunits. Second, the cross-linked hexamers were prevented from polymerizing further into hyperstable capsid-like structures by mutations (W184A and M185A) that interfered with dimeric association between the C-terminal domains that link adjacent hexamers. The structures of two different cross-linked CA hexamers were nearly identical, and we combined the non-mutated portions of the structures to generate an atomic resolution model for the native hexamer. This hybrid approach for structure determination should be applicable to other viral capsomers and protein–protein complexes in general.

Influence of intermolecular disulfide bonds on assembly of mouse MHC class I molecules (130.25)

Abstract Within the major histocompatibility (MHC) class I peptide-loading complex, tapasin and ERp57 are joined by a stable disulfide bond which is necessary for the efficient assembly of MHC class I molecules. In our investigation of murine tapasin, we identified a large covalently bound complex involving wild type mouse tapasin and ERp57. This complex, which migrates at ~150 kDa, is noticeably larger than the 110 kDa tapasin-ERp57 complex previously identified in human cells. Additional data suggest that the mouse MHC class I molecule Kd may be a part of this ~150 kDa complex. In order to investigate the importance of tapasin-ERp57 disulfide bonding for the assembly of murine MHC class I molecules, we introduced a tapasin C95S mutant into tapasin knockout mouse fibroblasts. Although this tapasin mutant is unable to disulfide bond with ERp57, mouse tapasin C95S was found to maintain a non-covalent association with ERp57. Mutation of tapasin C95 impaired the maturation and stability of MHC class I Kd molecules to a greater extent than was previously observed for the human MHC class I molecule, B*4402. Furthermore, the maturation and stability of mouse MHC class I Kb molecules was also impaired in cells expressing mouse tapasin C95S. In total, these results highlight inter-species differences in the MHC class I assembly pathway and emphasize the importance of studying diverse MHC class I allotypes.

Establishing regiocontrol of disulfide bond isomers of α‐conotoxin ImI via the synthesis of N‐to‐C cyclic analogs

alpha-Conotoxins are multiple disulfide bond containing peptides that are isolated from venomous marine cone snails. They display remarkable selectivity for different subtypes of nicotinic acetylcholine receptors (nAChRs). While alpha-conotoxins display poor resistance to in vivo degradation by proteases, which limits their use as drug leads, N-to-C cyclization via an oligopeptide spacer unit has been previously shown to improve stability. However, the effect of N-to-C cyclization on the formation of the disulfide bond framework is not fully understood. Four N-to-C cyclic analogs of alpha-conotoxin ImI; cImI-A, cImI-betaA, cImI-AG, and cImI-AGG were synthesized to evaluate the effect of oligopeptide spacer length on disulfide bond selectivity and stability to proteolysis. Different ratios of disulfide bond isomers were obtained for each analog using a nonselective random disulfide bond forming strategy, which was dependent on the length of the spacer. To identify each isomer obtained using the random strategy, and to gain access to disulfide bond isomers otherwise unattainable using the random strategy, both the native (globular) and ribbon isomers were synthesized in good yield and purity using a selective orthogonal cysteine protecting group strategy. As such, a random oxidation strategy showed a clear preference for the ribbon isomer in cImI-A. The cyclic globular isomers showed a high resistance to enzymatic degradation compared to the ribbon isomers, with the cImI-A and cImI-AG globular isomers demonstrating the highest stability. These results suggest that cyclization can improve the biochemical stability of conotoxins with potential applications in the development of drugs.

Effects of Hydroxylpropyl-β-Cyclodextrin on in Vitro Insulin Stability

The objective of this study was to elucidate the effects of hydroxylpropyl-β-cyclodextrin (HP-β-CD) on the in vitro stability of insulin. It was found that HP-β-CD had positive effects on the stability of insulin in acid and base and under high temperature conditions. Furthermore, use of HP-β-CD could also increase the stability of disulfide bonds which are important to the conformation of insulin. Through 1H-NMR experiments it was found that the protective effect of HP-β-CD was due to complexation with insulin. The results suggest that the presence of HP-β-CD could improve the stability of insulin in different environments.

Kinetic and Thermodynamic Aspects of Cellular Thiol–Disulfide Redox Regulation

Regulation of intracellular thiol-disulfide redox status is an essential part of cellular homeostasis. This involves the regulation of both oxidative and reductive pathways, production of oxidant scavengers and, importantly, the ability of cells to respond to changes in the redox environment. In the cytosol, regulatory disulfide bonds are typically formed in spite of the prevailing reducing conditions and may thereby function as redox switches. Such disulfide bonds are protected from enzymatic reduction by kinetic barriers and are thus allowed to exist long enough to elicit the signal. Factors that affect the rate of thiol-disulfide exchange and stability of disulfide bonds are discussed within the framework of the underlying chemical foundations. This includes the effect of thiol acidity (pK(a)), the local electrostatic environment, molecular strain, and entropy. Even though a thiol-disulfide exchange reaction is thermodynamically favorable, it will only take place if the activation energy to form the transition state complex can be overcome. This is accomplished by enzymes, such as the oxidoreductases, that direct reactions in thermodynamically favorable directions by decreasing the activation energy barrier.

Disulfide and multisulfide antitumor agents and their modes of action

A variety of natural products that contain disulfide or multisulfide bonds were found to display potent biological activities, including antitumor activities. At the center of these biological activities are disulfide or multisulfide moieties. The importance of disulfide or multisulfide groups in the areas of chemistry, biology, and pharmacology has been well recognized. Among these agents, especially noteworthy are mitomycin disulfides, leinamycin, thiarubrines, varacins, calicheamicins, and esperamicins. Their general features, including their biological profiles, peculiar structures, and related chemistries, were summarized and more importantly, their working mechanisms were elucidated in detail in this review. Mechanistic studies of these compounds have provided evidence of the key role of disulfide or multisulfide groups. In general, the cleavage of disulfide or multisulfide bonds produces thiol (or thiolate), which triggers an activation cascade leading to the generation of highly reactive electrophile(s) or cytotoxic species that may cause DNA strand scission. The main concerns with the mode of action are the reactivity and stability of disulfide and multisulfide bonds, their cleavage conditions, and the generation of toxic species. A range of studies for each agent was executed to gather important information on their activation, and the obtained information was gradually integrated to give some clues to the agents' working mechanisms. Such information may be further used to generate biomechanistically designed and more potent derivatives.

Effects of Mutations Involving Cysteine Residues Distal to the S281HCC Motif at the C-Terminus on the Functional Characteristics of a Truncated Ectodomain-Only Thyrotropin Receptor Anchored on Glycosylphosphatidyl-Inositol

Cysteine (Cys) residues pair to form disulfide bonds that are important in maintaining structure and function of the thyrotropin receptor (TSHR). There are 11 Cys residues in the ectodomain (ECD). Cys 41 at the N-terminus and Cys 283 at the SHCC motif have been identified as important for ligand binding. The present study evaluated the effects of mutating Cys distal to the S281HCC motif at the C-terminus of the ECD on the functional characteristics of TSHR. We introduced (i) individual Cys and (ii) consecutive cumulative Cys mutations into the starting template SHCS-TSHR, a truncated TSHR-ECD moiety previously shown to behave like the wild-type TSHR. Each mutant receptor was evaluated for relative specific binding (RSB), calculated as a measure of TSH-binding ability after normalization with receptor surface expression. In the first approach, RSB was severely affected when Cys 390 and Cys 398 were individually switched to serine. Failed receptor trafficking occurred with Cys 408 mutation. These findings were likely results of altered receptor conformation due to illegitimate disulfide bridge formation. Only SHCS-301 TSHR bound TSH in a specific manner, and it formed the base for sequential Cys mutations. Through this second approach, both Cys 301 and 390 could be removed simultaneously without hindering TSH binding significantly. Cys 398, however, was shown to be critical. Its absence resulted in huge loss of TSH binding. Leaving Cys 283 and 398 as the only Cys pair in the C-terminus alone could support 40% of the total ligand-binding capacity. From these data, we proposed Cys 398 as a stable disulfide bond partner of Cys 283, corroborating with a model based on evolutionary history of TSHR across species. This pairing of Cys 283 and Cys 398 also provides an objective alternative to conventional hypotheses on Cys coupling based on other predictive models.

S-Nitrosylation of Surfactant Protein-D Controls Inflammatory Function

The pulmonary collectins, surfactant proteins A and D (SP-A and SP-D) have been implicated in the regulation of the innate immune system within the lung. In particular, SP-D appears to have both pro- and anti-inflammatory signaling functions. At present, the molecular mechanisms involved in switching between these functions remain unclear. SP-D differs in its quaternary structure from SP-A and the other members of the collectin family, such as C1q, in that it forms large multimers held together by the N-terminal domain, rather than aligning the triple helix domains in the traditional “bunch of flowers” arrangement. There are two cysteine residues within the hydrophobic N terminus of SP-D that are critical for multimer assembly and have been proposed to be involved in stabilizing disulfide bonds. Here we show that these cysteines exist within the reduced state in dodecameric SP-D and form a specific target for S-nitrosylation both in vitro and by endogenous, pulmonary derived nitric oxide (NO) within a rodent acute lung injury model. S-nitrosylation is becoming increasingly recognized as an important post-translational modification with signaling consequences. The formation of S-nitrosothiol (SNO)-SP-D both in vivo and in vitro results in a disruption of SP-D multimers such that trimers become evident. SNO-SP-D but not SP-D, either dodecameric or trimeric, is chemoattractive for macrophages and induces p38 MAPK phosphorylation. The signaling capacity of SNO-SP-D appears to be mediated by binding to calreticulin/CD91. We propose that NO controls the dichotomous nature of this pulmonary collectin and that posttranslational modification by S-nitrosylation causes quaternary structural alterations in SP-D, causing it to switch its inflammatory signaling role. This represents new insight into both the regulation of protein function by S-nitrosylation and NO's role in innate immunity.

Conformational stability of disulfide bonds in redox processes

The protein sulfur functions have numerous cellular fundamental roles. They are in charge of the maintenance of the cellular reduction potential, which in turn governs gene expression. In addition, the redox cycling of disulfide/thiol functions has a great importance in the protein folding–unfolding process, which governs the functioning of enzymes. In our previous studies, we showed that the redox properties of disulfide bonds differ considerably. An analysis of the structure of the disulfide bonds in several proteins in the Protein Data Bank indicated that there are two types of conformations. Thus, we have compared the structures of the anions in these two types of conformation and the SS bond dissociation energy of the anion. For both conformations, the SS bond length of the anion and the electronic affinity are very close. However, the bond dissociation energy (BDE) varies.

Endoplasmic Reticulum Stress Increases the Expression of Methylenetetrahydrofolate Reductase through the IRE1 Transducer

Methylenetetrahydrofolate reductase (MTHFR), an enzyme in folate and homocysteine metabolism, influences many cellular processes including methionine and nucleotide synthesis, methylation reactions, and maintenance of homocysteine at nontoxic levels. Mild deficiency of MTHFR is common in many populations and modifies risk for several complex traits including vascular disease, birth defects, and cancer. We recently demonstrated that MTHFR can be up-regulated by NF-kappaB, an important mediator of cell survival that is activated by endoplasmic reticulum (ER) stress. This observation, coupled with the reports that homocysteine can induce ER stress, prompted us to examine the possible regulation of MTHFR by ER stress. We found that several well characterized stress inducers (tunicamycin, thapsigargin, and A23187) as well as homocysteine could increase Mthfr mRNA and protein in Neuro-2a cells. The induction of MTHFR was also observed after overexpression of inositol-requiring enzyme-1 (IRE1) and was inhibited by a dominant-negative mutant of IRE1. Because IRE1 triggers c-Jun signaling, we examined the possible involvement of c-Jun in up-regulation of MTHFR. Transfection of c-Jun and two activators of c-Jun (LiCl and sodium valproate) increased MTHFR expression, whereas a reported inhibitor of c-Jun (SP600125) and a dominant-negative derivative of c-Jun N-terminal kinase-1 reduced MTHFR activation. We conclude that ER stress increases MTHFR expression and that IRE1 and c-Jun mediate this activation. These findings provide a novel mechanism by which the ER can regulate homeostasis and allude to an important role for MTHFR in cell survival.

The Machinery for Oxidative Protein Folding in Thermophiles

Disulfide bonds are required for the stability and function of many proteins. A large number of thiol-disulfide oxidoreductases, belonging to the thioredoxin superfamily, catalyze protein disulfide bond formation in all living cells, from bacteria to humans. The protein disulfide isomerase (PDI) is the eukaryotic factor that catalyzes oxidative protein folding in the endoplasmic reticulum; by contrast, in prokaryotes, a family of disulfide bond (Dsb) proteins have an equivalent outcome in the bacterial periplasm. Recently the results from genome analysis suggested an important role for disulfide bonds in the structural stabilization of intracellular proteins from thermophiles. A specific protein disulfide oxidoreductase (PDO) has a key role in intracellular disulfide shuffling in thermophiles. Here we focus on the structural and functional characterization of PDO correlated with the multifunctional eukaryotic PDI. In addition, we highlight the chimeric nature of the machinery for oxidative protein folding in thermophiles in comparison with the mesophilic bacterial and eukaryal counterparts.