Structural and functional studies of suppressor of copper sensitivity proteins from Proteus mirabilis

pSSbond-PseAAC: Prediction of disulfide bonding sites by integration of PseAAC and statistical moments

Disulfide bonds occurred in majority of secreted protein. Formation of correct disulfide bonds are must for achieving native conformation, solubility and activity. Production of recombinant proteins containing disulfide bond for therapeutic, diagnostic and various other purposes is a challenging task of research. Production of such proteins in the reducing cytosolic compartment of E. coli usually ends up in inclusion bodies formation. Refolding of inclusion bodies can be difficult, time and labor consuming and uneconomical. Translocation of these proteins into the oxidative periplasmic compartment provides correct environment to undergo proper disulfide bonds formation and thus achieving native conformation. However, not all proteins can be efficiently translocated to the periplasm with the help of bacterial signal peptides. Therefore, fusion to a small well-folded and stable periplasmic protein is more promising for periplasmic production of disulfide bonded proteins. In the past decades, several full-length proteins or domains were used for enhancing translocation and solubility. Here, protein fusion tags that significantly increase the yields of target proteins in the periplasmic space are reviewed.

The insulin superfamily of peptides is ubiquitous within vertebrates and invertebrates and is characterized by the presence of a set of three disulfide bonds in a unique disposition. With the exception of insulin-like growth factors I and II, which are single chain peptides, the remaining 8 members of the human insulin superfamily are two-chain peptides containing one intramolecular and two intermolecular disulfide bridges. These structural features have long made the chemical synthesis of the peptides a considerable challenge, in particular, including their correct disulfide bond pairing and formation. However, they have also afforded the opportunity to develop modern solid phase synthesis methods for the preparation of such peptides that incorporate novel or improved chemical methods for the controlled introduction of both disulfide bonds and their surrogates, both during and after peptide chain assembly. In turn, this has enabled a detailed probing of the structure and function relationship of this small but complex superfamily of peptides. After initially using and subsequently identifying significant limitations of the approach of simultaneous random chain combination and oxidative folding, our laboratory undertook to develop robust chemical synthesis strategies in concert with orthogonal cysteine S-protecting groups and corresponding regioselective disulfide bond formation. These have included the separate synthesis of each of the two chains or of the two chains linked by an artificial C-peptide that is removed following postoxidative folding. These, in turn, have enabled an increased ease of acquisition in a good yield of not only members of human insulin superfamily but other insulin-like peptides. Importantly, these successful methods have enabled, for the first time, a detailed analysis of the role that the disulfide bonds play in the structure and function of such peptides. This was achieved by selective removal of the disulfide bonds or by the judicious insertion of disulfide isosteres that possess structurally subtle variations in bond length, hydrophobicity, and angle. These include lactam, dicarba, and cystathionine, each of which has required modifications to the peptide synthesis protocols for their successful placement within the peptides. Together, these synthesis improvements and the novel chemical developments of cysteine/cystine analogues have greatly aided in the development of novel insulin-like peptide (INSL) analogues, principally with intra-A-chain disulfide isosteres, possessing not only improved functional properties such as increased receptor selectivity but also, with one important and unexpected exception, greater in vivo half-lives due to stability against disulfide reductases. Such analogues greatly will aid further biochemical and pharmacological analyses to delineate the structure-function relationships of INSLs and also future potential drug development.

The hydroxamate siderophore receptor FhuA is a TonB-dependent outer membrane protein of Escherichia coli composed of a C-terminal 22-stranded beta-barrel occluded by an N-terminal globular cork domain. During siderophore transport into the periplasm, the FhuA cork domain has been proposed to undergo conformational changes that allow transport through the barrel lumen; alternatively, the cork may be completely displaced from the barrel. To probe such changes, site-directed cysteine mutants in the cork domain (L109C and Q112C) and in the barrel domain (S356C and M383C) were created within the putative siderophore transport pathway. Molecular modeling predicted that the double cysteine mutants L109C/S356C and Q112C/M383C would form disulfide bonds, thereby tethering the cork and barrel domains. The double cysteine FhuA mutants were denatured under nonreducing conditions and fluorescently labeled with thiol-specific Oregon Green maleimide. Subsequent SDS-PAGE analysis revealed two distinct species: FhuA containing a disulfide bond and FhuA with free sulfhydryl groups. To address the role of the putative siderophore transport pathway and to evaluate possible rearrangements of the cork domain during ferricrocin transport, disulfide bond formation was enhanced by an oxidative catalyst. Cells containing double cysteine FhuA mutants that were subjected to oxidation during ferricrocin transport exhibited disulfide bond formation to near completion. After disulfide tethering of the cork to the barrel, ferricrocin transport was equivalent to transport by untreated cells. These results demonstrate that blocking the putative siderophore transport pathway does not abrogate ferricrocin uptake. We propose that, during siderophore transport through FhuA, the cork domain remains within the barrel rather than being displaced.

A fluorescent-based HPLC assay for quantification of cysteine and cysteamine adducts in Escherichia coli-derived proteins

Redox Regulation Facilitates Optimal Peptide Selection by MHC Class I during Antigen Processing

We have used total chemical synthesis to prepare [V15A]crambin-alpha-carboxamide, a unique protein analogue that eliminates a salt bridge between the delta-guanidinium of the Arg(10) side chain and the alpha-carboxylate of Asn(46) at the C-terminus of the polypeptide chain. This salt bridge is thought to be important for the folding and stability of the crambin protein molecule. Folding, with concomitant disulfide bond formation, of the fully reduced [V15A]crambin-alpha-carboxamide polypeptide was less efficient than folding/disulfide formation for the [V15A]crambin polypeptide under a standard set of conditions. To probe the origin of this less efficient folding/disulfide bond formation, we separately crystallized purified synthetic [V15A]crambin-alpha-carboxamide and chemically synthesized [V15A]crambin and solved their X-ray structures. The crystal structure of [V15A]crambin-alpha-carboxamide showed that elimination of the Arg(10)-Asn(46) salt bridge caused disorder of the C-terminal region of the polypeptide chain and affected the overall 'tightness' of the structure of the protein molecule. These studies, enabled by chemical protein synthesis, strongly suggest that in native crambin the Arg(10)-Asn(46) salt bridge contributes to efficient formation of correct disulfide bonds and also to the well-ordered structure of the protein molecule.

We have previously demonstrated that the coated vesicle vacuolar H(+)-ATPase (V-ATPase) can be inactivated by formation of intramolecular disulfide bonds (Feng, Y., and Forgac, M. (1992) J. Biol. Chem. 267, 19769-19772). The disulfide bond responsible for inactivation can be distinguished from other disulfide bonds that form by the fact that formation of the inactivating disulfide bond is blocked by ATP or high ionic strength. By taking advantage of these properties, we selectively labeled the ATPase at the relevant cysteine residues with fluorescein maleimide. After analyzing the proteolytic fragments that contain the labeled cysteine residues, we found that cysteine 254 and cysteine 532 in subunit A of the bovine V-ATPase are the residues that form the disulfide bond resulting in inactivation of the enzyme. Cysteine 254 and cysteine 532 correspond to 2 of the 3 cysteine residues that are conserved in all available V-ATPase A subunit sequences. Cysteine 254 is located in the consensus motif, G(X)4GKT, corresponding to residues 250-257, which is conserved in many nucleotide binding proteins. Cysteine 532 is located in a region not previously shown to be in proximity to the nucleotide binding site. Modification of cysteine 254 by disulfide bond formation with cysteine 532 or thio-disulfide exchange with cystine does not impair binding of 2-azido-[32P]ATP to the A subunit. The inhibition is therefore likely caused by disruption of the catalytic function of the ATPase on formation of the disulfide bond. A possible role in regulating intracellular acidification by reversible sulfhydryl oxidation and reduction is discussed.

The DsbA-DsbB system affects the formation of disulfide bonds in periplasmic but not in intramembraneous protein domains

It is widely accepted, although never demonstrated, that the formation of a disulfide bond in the majority of immunoglobulin (Ig)-like domains stabilizes their final conformation and thus is essential to their functioning as adhesion/recognition molecules. The myelin P0 protein, which has been shown directly to behave as a homophilic adhesion molecule, contains a single Ig-like domain, stabilized by a putative Cys21-Cys98 disulfide bond. To test if this bond is indeed necessary to the adhesive function of P0, the nucleotides in the P0 cDNA coding for Cys21 were altered to code for an alanine. The mutated P0 cDNA was transfected into Chinese hamster ovary cells, expression of the mutated P0 protein was characterized, and the adhesiveness of Cys21-mutated P0-expressing cells and that of cells expressing equivalent surface amounts of the unmutated protein were compared. It was found, as we previously reported, that incubation of a single cell suspension of the unmutated P0-expressing cells resulted in the rapid formation of large aggregates. In contrast, after a similar incubation the cells expressing the Cys21-mutated P0 were still mostly single cells, a result indistinguishable from that observed with the control transfected cells. This suggests that the P0 protein, when mutated at Cys21, does not behave as a homophilic adhesion molecule, which in turn implies that the formation of an Ig domain disulfide bond is essential to the functioning of this molecule.

MAPK are activated by and orchestrate responses to multiple, diverse stimuli. Although these responses involve the increased phosphorylation of substrate effector proteins, e.g. transcription factors, the mechanisms by which responses are tailored to particular stimuli are unclear. In the fission yeast Schizosaccharomyces pombe, the Sty1 MAPK is crucial for changes in gene expression that allow adaptation to many forms of environmental stress. Here, we have identified two cysteine residues in Sty1, Cys-153 and Cys-158, that are important for hydrogen peroxide-induced gene expression and oxidative stress resistance but not for other functions of Sty1. Many Sty1-dependent changes in gene expression are mediated by the Atf1 transcription factor. In response to stress, Sty1 increases Atf1 levels by (i) promoting increases in atf1 mRNA and by (ii) directly phosphorylating and stabilizing Atf1 protein. Although dispensable for phosphorylation and stabilization of Atf1 protein, we find that both Cys-153 and Cys-158 are required for increases in atf1 mRNA levels and Atf1-dependent gene expression in response to hydrogen peroxide but not osmotic stress. Indeed, our data indicate that oxidation of Sty1, by formation of a disulfide bond between Cys-153 and Cys-158, is important for maintaining atf1 mRNA stability at high concentrations of hydrogen peroxide. Together, these data reveal that redox regulation of cysteine thiols in Sty1 is involved in a stress-specific mechanism regulating transcriptional responses to oxidative stress. Intriguingly, the conservation of these cysteine residues in other MAPK raises the possibility that similar mechanisms may ensure appropriate responses to hydrogen peroxide in other eukaryotes.

The ATP-binding cassette (ABC) transporter ABCB6 is a mitochondrial porphyrin transporter that activates porphyrin biosynthesis. ABCB6 lacks a canonical mitochondrial targeting sequence but reportedly traffics to other cellular compartments such as the plasma membrane. How ABCB6 reaches these destinations is unknown. In this study, we show that endogenous ABCB6 is glycosylated in multiple cell types, indicating trafficking through the endoplasmic reticulum (ER), and has only one atypical site for glycosylation (NXC) in its amino terminus. ABCB6 remained glycosylated when the highly conserved cysteine (Cys-8) was substituted with serine to make a consensus site, NXS. However, this substitution blocked ER exit and produced ABCB6 degradation, which was mostly reversed by the proteasomal inhibitor MG132. The amino terminus of ABCB6 has an additional highly conserved ER luminal cysteine (Cys-26). When Cys-26 was mutated alone or in combination with Cys-8, it also resulted in instability and ER retention. Further analysis revealed that these two cysteines form a disulfide bond. We discovered that other ABC transporters with an amino terminus in the ER had similarly configured conserved cysteines. This analysis led to the discovery of a disease-causing mutation in the sulfonylurea receptor 1 (SUR1)/ABCC8 from a patient with hyperinsulinemic hypoglycemia. The mutant allele only contains a mutation in a conserved amino-terminal cysteine, producing SUR1 that fails to reach the cell surface. These results suggest that for ABC transporters the propensity to form a disulfide bond in the ER defines a unique checkpoint that determines whether a protein is ER-retained.

A newly discovered teleost disulfide isomerase, thioredoxin domain containing 5 (TXNDC5), from big-belly seahorse (Hippocampus abdominalis): Insights into its molecular and functional properties and immune regulatory functions

Redox reactions involving cysteine thiol-disulfide exchange are crucial for the intracellular monitoring of hydrogen peroxide (H(2)O(2)). Yap1, the master transcription factor for the oxidative stress response in budding yeast, is activated by the formation of disulfide bonds in response to H(2)O(2). Gpx3 (glutathione peroxidase-like protein 3) acts as a receptor for H(2)O(2), and Ybp1 (Yap1-binding protein 1) is crucial for Gpx3-dependent disulfide bond formation in Yap1. We previously reported that Tsa1, a major peroxiredoxin in yeast cells, is required for activation of Yap1 in a widely used yeast strain, W303-1b, carrying the ybp1-1 mutant allele encoding a truncated Ybp1 protein. In the present study, we show that Tsa1 can interact with Yap1 via disulfide linkages and induce the formation of intramolecular disulfide bonds in Yap1 in ybp1-1 cells. The results provide evidence that Prx can have intrinsic activity as an H(2)O(2) receptor and can relay H(2)O(2) as a signal to the Prx target proteins in terms of formation of disulfide linkage. Furthermore, our data reveal that there is more of the reduction-resistant active form of Yap1 (i.e. Yap1 (oxII)) when it is partnered with Gpx3 than with Tsa1. These data support our hypothesis that changes in the redox status of Yap1 to reduction-resistant forms by multiple disulfide bond formation are important for determining the level and duration of Yap1 activity in the dynamic equilibrium of redox reactions in cells exposed to H(2)O(2).

Background: Congenital hypothyroidism (CH) is caused by mutations in cysteine residues, including Cys655 and Cys825 that form disulfide bonds in thyroid peroxidase (TPO). It is highly likely that these disulfide bonds could play an important role in TPO activity. However, to date, no study has comprehensively analyzed cysteine mutations that form disulfide bonds in TPO. In this study, we induced mutations in cysteine residues involved in disulfide bonds formation and analyzed their effect on subcellular localization, degradation, and enzyme activities to evaluate the importance of disulfide bonds in TPO activity. Methods: Vector plasmid TPO mutants, C655F and C825R, known to occur in CH, were transfected into HEK293 cells. TPO activity and protein expression levels were measured by the Amplex red assay and Western blotting. The same procedure was performed in the presence of MG132 proteasome inhibitor. Subcellular localization was determined using immunocytochemistry and flow cytometry. The locations of all disulfide bonds within TPO were predicted using in silico analysis. All TPO mutations associated with disulfide bonds were induced. TPO activity and protein expression levels were also measured in all TPO mutants associated with disulfide bonds using the Amplex red assay and Western blotting. Results: C655F and C825R showed significantly decreased activity and protein expression compared with the wild type (WT) (p < 0.05). In the presence of the MG132 proteasome inhibitor, the protein expression level of TPO increased to a level comparable with that of the WT without increases in its activity. The degree of subcellular distribution of TPO to the cell surface in the mutants was lower compared with the WT TPO. Twenty-four cysteine residues were involved in the formation of 12 disulfide bonds in TPO. All TPO mutants harboring an amino acid substitution in each cysteine showed significantly reduced TPO activity and protein expression levels. Furthermore, the differences in TPO activity depended on the position of the disulfide bond. Conclusions: All 12 disulfide bonds play an important role in the activity of TPO. Furthermore, the mutations lead to misfolding, degradation, and membrane insertion.