Effect of precipitated morphine withdrawal on post-translational processing of prothyrotropin releasing hormone (proTRH) in the ventrolateral column of the midbrain periaqueductal gray

We have demonstrated that during opiate withdrawal, preprothyrotropin releasing hormone (preproTRH) mRNA is increased in neurons of the midbrain periaqueductal gray matter (PAG) while the concentration of TRH remained unaltered, suggesting that the processing of proTRH may be different in this region of the brain. The aim of the present study was to determine which of the proTRH-derived peptides are affected by opiate withdrawal in the PAG. These changes were compared to other TRH-containing areas such as the hypothalamic paraventricular nucleus (PVN), median eminence (ME) and the lateral hypothalamus (LH). Control and morphine-treated rats 24 h following naltrexone-precipitated withdrawal were decapitated and the brain microdissected. Pooled samples from each animal group were acid extracted, and peptides were electrophoretically separated then analyzed by specific radioimmunoassay. Opiate withdrawal caused a significant change in the level of some post-translational processing products derived from the TRH precursor. In the PAG, opiate withdrawal resulted in an accumulation of the intervening preproTRH(83-106) peptide from the N-terminal side of the prohormone, while the levels of the C-terminal preproTRH(208-285) peptide were reduced, with no change in preproTRH(25-50) or TRH, itself, as compared to control animals. Immunohistochemical analysis also showed significant increases in cellular preproTRH(83-106) peptide immunolabeling in the PAG. Opiate withdrawal in the lateral hypothalamus, unlike from the PAG, was accompanied by an increase in the concentration of TRH. In addition, western blot analysis showed that during opiate withdrawal, the mature form of the prohormone convertase 2 (PC2) increased only in PAG as compared with their respective controls. Thus, these results demonstrate a region-specific regulation of TRH prohormone processing in the brain, which may engage PC2, further suggesting a role for specific proTRH-derived peptides in the manifestations of opiate withdrawal.     

The disulfide bond formation (Dsb) system

In oxidative folding of proteins in the bacterial periplasmic space, disulfide bonds are introduced by the oxidation system and isomerized by the reduction system. These systems utilize the oxidizing and the reducing equivalents of quinone and NADPH, respectively, that are transmitted across the cytoplasmic membrane through integral membrane components DsbB and DsbD. In both pathways, alternating interactions between a Cys-XX-Cys-containing thioredoxin domain and other regulatory domain lead to the maintenance of oxidized and reduced states of the specific terminal enzymes, DsbA that oxidizes target cysteines and DsbC that reduces an incorrect disulfide to allow its isomerization into the physiological one. Molecular details of these remarkable biochemical cascades are being rapidly unraveled by genetic, biochemical, and structural analyses in recent years.     

Statistics of disulfide bond formation in proteins

A new set of statistical expressions describing the reformation of disulfide bonds from SH groups is proposed. The results of the statistical calculations of disulfide bond reformation are discussed in terms of protein folding.     

Monitoring disulfide bond formation in the eukaryotic cytosol

Glutathione is the most abundant low molecular weight thiol in the eukaryotic cytosol. The compartment-specific ratio and absolute concentrations of reduced and oxidized glutathione (GSH and GSSG, respectively) are, however, not easily determined. Here, we present a glutathione-specific green fluorescent protein-based redox probe termed redox sensitive YFP (rxYFP). Using yeast with genetically manipulated GSSG levels, we find that rxYFP equilibrates with the cytosolic glutathione redox buffer. Furthermore, in vivo and in vitro data show the equilibration to be catalyzed by glutaredoxins and that conditions of high intracellular GSSG confer to these a new role as dithiol oxidases. For the first time a genetically encoded probe is used to determine the redox potential specifically of cytosolic glutathione. We find it to be -289 mV, indicating that the glutathione redox status is highly reducing and corresponds to a cytosolic GSSG level in the low micromolar range. Even under these conditions a significant fraction of rxYFP is oxidized.     

Human pancreas-specific protein disulfide isomerase homolog (PDIp) is redox-regulated through formation of an inter-subunit disulfide bond

Protein disulfide isomerase (PDI) and its homologs are catalysts of the formation of disulfide bonds in secretory proteins, and they also serve as molecular chaperones. In the present study, we investigated the redox-mediated regulation of the structures and functions of human pancreas-specific PDI homolog (PDIp). We found that formation of an inter-subunit disulfide bond in the recombinant human PDIp can alter not only its structure, but also its functions. PDIp exists predominantly as monomer under reducing conditions, but the dimeric form is significantly increased following the removal of the reducing agent, due to the formation of an inter-subunit disulfide bond. The oxidized PDIp (with an inter-subunit disulfide bond) appears to expose more hydrophobic patches and is more sensitive to protease digestion compared to the reduced form. Along with these structural changes, the oxidized PDIp also exhibits an enhanced chaperone activity. The formation of the inter-subunit disulfide bond in PDIp is mainly contributed by its non-active cysteine residue (cysteine-4), which is only present in human and primate PDIp, but not in rodent PDIp. In addition, we observed that the formation of the inter-subunit disulfide bond in PDIp is redox-dependent and is favored under oxidizing conditions, and that PDIp can function as a chaperone to form stable complexes with various non-native cellular proteins, particularly under oxidizing conditions. In light of these observations, it is concluded that the structures and functions of human PDIp are redox-regulated through formation of an inter-subunit disulfide bond between two cysteine-4 residues.     

Disruption of disulfide bonds exhibits differential effects on trafficking of regulated secretory proteins

For several secretory proteins, it has beenhypothesized that disulfide-bonded loop structures are required forsorting to secretory granules. To explore this hypothesis, we employeddithiothreitol (DTT) treatment in live pancreatic islets, as well as inPC-12 andGH₄C₁cells. In islets, disulfide reduction in the distal secretory pathwaydid not increase constitutive or constitutive-like secretion ofproinsulin (or insulin). In PC-12 cells, DTT treatment caused adramatic increase in unstimulated secretion of newly synthesizedchromogranin B (CgB), presumably as a consequence of reducing thesingle conserved chromogranin disulfide bond (E. Chanat, U. Weiss, W. B. Huttner, and S. A. Tooze. EMBO J. 12: 2159-2168, 1993). However, inGH₄C₁cells that also synthesize CgB endogenously, DTT treatment reducednewly synthesized prolactin and blocked its export, whereas newlysynthesized CgB was routed normally to secretory granules. Moreover, ontransient expression inGH₄C₁cells, CgA and a CgA mutant lacking the conserved disulfide bond showedcomparable multimeric aggregation properties and targeting to secretorygranules, as measured by stimulated secretion assays. Thus theconformational perturbation of regulated secretory proteins caused bydisulfide disruption leads to consequences in protein trafficking thatare both protein and cell type dependent.

     

Use of carboethoxysulfenyl chloride for disulfide bond formation

The use of carboethoxysulfenyl chloride for disulfide bond formation and concomitant cyclization of five peptides was investigated. Even though cyclic peptides were obtained very rapidly and in good yields when cyclization was performed in aqueous media at different pHs (4 to 7), the final crude peptides were found to contain closely related impurities which, in the case of somatostatin and pressinoic acid, were not generated by air oxidation. This observation may limit the use of carboethoxysulfenyl chloride to those cases where other methods of disulfide bond formation prove inadequate.     

Pathways of disulfide bond formation in Escherichia coli

Disulfide bond formation is required for the correct folding of many secreted proteins. Cells possess protein-folding catalysts to ensure that the correct pairs of cysteine residues are joined during the folding process. These enzymatic systems are located in the endoplasmic reticulum of eukaryotes or in the periplasm of Gram-negative bacteria. This review focuses on the pathways of disulfide bond formation and isomerization in bacteria, taking Escherichia coli as a model.     

Amyloidogenic synthetic peptides of beta2-microglobulin--a role of the disulfide bond

To search for the essential regions responsible for the beta2-microglobulin (beta2-m) amyloid fibril formation, we synthesized six peptides corresponding to six of the seven beta-sheets in the native structure of beta2-m, and examined their amyloidogenicity. Among the peptides examined, peptide (21-31) (strand B) and the mixture of peptide (21-31) and (78-86) (strand F) showed fibril formation at both pH 2.5 and 7.5. Peptide (21-31) is the N-terminal half of the previously reported proteolytic fragment of beta2-m, Ser21-Lys41 (K3), suggesting that this region may be the essential core. Interestingly, the dimer formation of peptide (21-31) by the disulfide bond substantially facilitated the fibril formation, indicating that the disulfide bond is important for the structural stability of the fibrils.     

Disruption of microfilaments alters laminin synthesis but not laminin trafficking in NHEKin vitro

Summary Laminin synthesis and deposition are concomitant with the development of a basal lamina between the human epidermis and the underlying dermis. One of the challenges in tissue engineering of human epidermal models is to develop substrates and conditions that encourage the development of a basement membrane. The purpose of this study was to determine if actin filaments and/or microtubules are involved in the synthesis/secretion of laminin by normal human epidermal keratinocytes (NHEK)in vitro. NHEK synthesize and secrete laminin subunits B1, B2, and M but little, if any, of laminin subunit A. Data indicate that disruption of microfilaments by the destabilizing agent, cytochalasin D, had no apparent effect on the relative synthesis rates of most cytosolic proteins as, revealed by one-dimensional sodium dodecyl sulfate (SDS) gel electrophoresis. This drug, however, increased laminin B2 synthesis several fold over untreated controls. This enhanced synthetic rate was independent of the type of collagen, matrix on which the NHEK were grown. Similar increases in synthesis of the M and B1 laminin chains were not observed. To determine if this increase in synthesis lead to increases in laminin B2 secretion, laminin B2 was immunoprecipitated from both the apical and basal domains of NHEK cells grown on microporous membranes. While more laminin B1, B2, and M were secreted basally than apically, an observation consistent with laminin’s role in basal lamina formation, cytochalasin D had no apparent effect on either basal or apical laminin B2 secretion. Experiments with the microtubule destabilizer, nocodazole, showed no similar effects on laminin synthesis and/or secretion. We conclude that (a) disruption of the actin network in NHEK selectively increases the synthesis of laminin B2, (b) the secretion of laminin B2 from NHEK cells is not governed by either the microfilamentous cytoskeleton or the amount of laminin synthesized by NHEK, and (c) disruption of the microtubular network does not alter laminin synthesis or secretion.     

Protein disulfide bond formation in the cytoplasm during oxidative stress

The majority of disulfide-linked cytosolic proteins are thought to be enzymes that transiently form disulfide bonds while catalyzing oxidation-reduction (redox) processes. Recent evidence indicates that reactive oxygen species can act as signaling molecules by promoting the formation of disulfide bonds within or between select redox-sensitive proteins. However, few studies have attempted to examine global changes in disulfide bond formation following reactive oxygen species exposure. Here we isolate and identify disulfide-bonded proteins (DSBP) in a mammalian neuronal cell line (HT22) exposed to various oxidative insults by sequential nonreducing/reducing two-dimensional SDS-PAGE combined with mass spectrometry. By using this strategy, several known cytosolic DSBP, such as peroxiredoxins, thioredoxin reductase, nucleoside-diphosphate kinase, and ribonucleotide-diphosphate reductase, were identified. Unexpectedly, a large number of previously unknown DSBP were also found, including those involved in molecular chaperoning, translation, glycolysis, cytoskeletal structure, cell growth, and signal transduction. Treatment of cells with a wide range of hydrogen peroxide concentrations either promoted or inhibited disulfide bonding of select DSBP in a concentration-dependent manner. Decreasing the ratio of reduced to oxidized glutathione also promoted select disulfide bond formation within proteins from cytoplasmic extracts. In addition, an epitope-tagged version of the molecular chaperone HSP70 forms mixed disulfides with both beta4-spectrin and adenomatous polyposis coli protein in the cytosol. Our findings indicate that disulfide bond formation within families of cytoplasmic proteins is dependent on the nature of the oxidative insult and may provide a common mechanism used to control multiple physiological processes.     

Preparation of protein conjugates via intermolecular disulfide bond formation

Conjugates of two unlike proteins can be prepared via the intermolecular disulfide interchange reaction, namely, protein A containing thiol groups reacts with protein B containing 4-dithiopyridyl groups to yield a conjugate with the release of 4-thiopyridone. Thiol groups can be introduced into proteins upon amidination with methyl 3-mercaptopropionimidate ester or 2-iminothiolane, and 4-dithiopyridyl groups can be introduced into proteins with these same reagents in the presence of 4,4'-dithiodipyridine. 2-Iminothiolane is stable on storage in contrast to the known lability of imidate esters; therefore 2-iminothiolane is a more convenient reagent for the modification of protein than are the imidate esters. All the reactions can be carried out easily under mild conditions in good yields. Conjugates of bovine plasma albumin with itself, ribonuclease, or a copolymer of D-glutamic acid and D-lysine and of sheep antibody and horseradish peroxidase were prepared with modified proteins containing an average of 1 to 5 thiol or dithiopyridyl groups per mol. These conjugates formed mainly dimers, trimers, and tetramers. The peroxidase labeled antibody retained more than 80% of its enzymatic and antigenic binding activities.     

Characterization of the menaquinone-dependent disulfide bond formation pathway of Escherichia coli

In the protein disulfide-introducing system of Escherichia coli, plasma membrane-integrated DsbB oxidizes periplasmic DsbA, the primary disulfide donor. Whereas the DsbA-DsbB system utilizes the oxidizing power of ubiquinone (UQ) under aerobic conditions, menaquinone (MK) is believed to function as an immediate electron acceptor under anaerobic conditions. Here, we characterized MK reactivities with DsbB. In the absence of UQ, DsbB was complexed with MK8 in the cell. In vitro studies showed that, by binding to DsbB in a manner competitive with UQ, MK specifically oxidized Cys41 and Cys44 of DsbB and activated its catalytic function to oxidize reduced DsbA. In contrast, menadione used in earlier studies proved to be a more nonspecific oxidant of DsbB. During catalysis, MK8 underwent a spectroscopic transition to develop a visible violet color (lambdamax = 550 nm), which required a reduced state of Cys44 as shown previously for UQ color development (lambdamax = 500 nm) on DsbB. In an in vitro reaction system of MK8-dependent oxidation of DsbA at 30 degrees C, two reaction components were observed, one completing within minutes and the other taking >1 h. Both of these reaction modes were accompanied by the transition state of MK, for which the slower reaction proceeded through the disulfide-linked DsbA-DsbB(MK) intermediate. The MK-dependent pathway provides opportunities to further dissect the quinone-dependent DsbA-DsbB redox reactions.     

Generation of two isomers with the same disulfide connectivity during disulfide bond formation of human uroguanylin

Summary In a two-step selective disulfide-bond-forming reaction of human uroguanylin, a 16-residue peptide with two intramolecular disulfide bonds, two compounds (I and II) were formed, which could be detected by RP-HPLC after the second disulfide-bond-forming reaction and were isolated as single entities. Their primary structures, molecular weights, and disulfide connectivities proved to be identical, but their optical rotation values were different, suggesting that they are topological isomers. Only compound I was found to increase the cGMP levels in cultured T84 cells significantly. The ratio of these compounds was affected by the order of the disulfide-bond-forming reactions, but not by the solvent used. The presence of a carboxyl-terminal leucine residue seems to be crucial for stabilizing the conformation of the two isomers.     

HLA-B27 misfolding is associated with aberrant intermolecular disulfide bond formation (dimerization) in the endoplasmic reticulum

The class I protein HLA-B27 confers susceptibility to inflammatory arthritis in humans and when overexpressed in rodents for reasons that remain unclear. We demonstrated previously that HLA-B27 heavy chains (HC) undergo endoplasmic reticulum (ER)-associated degradation. We report here that HLA-B27 HC also forms two types of aberrant disulfide-linked complexes (dimers) during the folding and assembly process that can be distinguished by conformation-sensitive antibodies W6/32 and HC10. HC10-reactive dimers form immediately after HC synthesis in the ER and constitute at least 25% of the HC pool, whereas W6/32-reactive dimers appear several hours later and represent less than 10% of the folded HC. HC10-reactive dimers accumulate in the absence of tapasin or beta(2)-microglobulin, whereas W6/32-reactive dimers are not detected. Efficient formation of W6/32-reactive dimers appears to depend on the transporter associated with antigen processing, tapasin, and beta(2)-microglobulin. The unpaired Cys(67) and residues at the base of the B pocket that dramatically impair HLA-B27 HC folding are critical for the formation of HC10-reactive ER dimers. Although certain other alleles also form dimers late in the assembly pathway, ER dimerization of HLA-B27 may be unique. These results demonstrate that residues comprising the HLA-B27 B pocket result in aberrant HC folding and disulfide bond formation, and thus confer unusual properties on this molecule that are unrelated to peptide selection per se, yet may be important in disease pathogenesis.     

Sulfhydryl oxidase from egg white. A facile catalyst for disulfide bond formation in proteins and peptides.

Both metalloprotein and flavin-linked sulfhydryl oxidases catalyze the oxidation of thiols to disulfides with the reduction of oxygen to hydrogen peroxide. Despite earlier suggestions for a role in protein disulfide bond formation, these enzymes have received comparatively little general attention. Chicken egg white sulfhydryl oxidase utilizes an internal redox-active cystine bridge and a FAD moiety in the oxidation of a range of small molecular weight thiols such as glutathione, cysteine, and dithiothreitol. The oxidase is shown here to exhibit a high catalytic activity toward a range of reduced peptides and proteins including insulin A and B chains, lysozyme, ovalbumin, riboflavin-binding protein, and RNase. Catalytic efficiencies are up to 100-fold higher than for reduced glutathione, with typical K(m) values of about 110-330 microM/protein thiol, compared with 20 mM for glutathione. RNase activity is not significantly recovered when the cysteine residues are rapidly oxidized by sulfhydryl oxidase, but activity is efficiently restored when protein disulfide isomerase is also present. Sulfhydryl oxidase can also oxidize reduced protein disulfide isomerase directly. These data show that sulfhydryl oxidase and protein disulfide isomerase can cooperate in vitro in the generation and rearrangement of native disulfide pairings. A possible role for the oxidase in the protein secretory pathway in vivo is discussed.     

Periplasmic transit and disulfide bond formation of the autotransported Shigella protein IcsA

The Shigella outer membrane protein IcsA belongs to the family of type V secreted (autotransported) virulence factors. Members of this family mediate their own translocation across the bacterial outer membrane: the carboxy-terminal beta domain forms a beta barrel channel in the outer membrane through which the amino-terminal alpha domain passes. IcsA, which is localized at one pole of the bacterium, mediates actin assembly by Shigella, which is essential for bacterial intracellular movement and intercellular dissemination. Here, we characterize the transit of IcsA across the periplasm during its secretion. We show that an insertion in the dsbB gene, whose gene product mediates disulfide bond formation of many periplasmic intermediates, does not affect the surface expression or unipolar targeting of IcsA. However, IcsA forms one disulfide bond in the periplasm in a DsbA/DsbB-dependent fashion. Furthermore, cellular fractionation studies reveal that IcsA has a transient soluble periplasmic intermediate. Our data also suggest that IcsA is folded in a proteinase K-resistant state in the periplasm. From these data, we propose a novel model for the secretion of IcsA that may be applicable to other autotransported proteins.     

On the requirement of oxidizing reagent for the formation of a disulfide bond

Cyclization of a peptide through the formation of a disulfide bond between the SH groups of cysteines on the N- and C-terminals of peptide was studied in degassed water solution under vacuum. Cyclization went to completion although the solution was oxygen deficient (the number of oxygen molecules available for the reaction was at least 16 times less than the number of peptide molecules). This result indicates that, contrary to the common assumption, disulfide bond formation does not necessarily require an oxidant (O(2), I(2), etc.) to occur.