Graph-representation of oxidative folding pathways

Background  

The process of oxidative folding combines the formation of native disulfide bond with conformational folding resulting in the native three-dimensional fold. Oxidative folding pathways can be described in terms of disulfide intermediate species (DIS) which can also be isolated and characterized. Each DIS corresponds to a family of folding states (conformations) that the given DIS can adopt in three dimensions.     

Linear array of conserved sequence motifs to discriminate protein subfamilies: study on pyridine nucleotide-disulfide reductases

Background  

The pyridine nucleotide disulfide reductase (PNDR) is a large and heterogeneous protein family divided into two classes (I and II), which reflect the divergent evolution of its characteristic disulfide redox active site. However, not all the PNDR members fit into these categories and this suggests the need of further studies to achieve a more comprehensive classification of this complex family.     

Disruption of reducing pathways is not essential for efficient disulfide bond formation in the cytoplasm of <Emphasis Type="Italic">E. coli</Emphasis>

Background  

The formation of native disulfide bonds is a complex and essential post-translational modification for many proteins. The large scale production of these proteins can be difficult and depends on targeting the protein to a compartment in which disulfide bond formation naturally occurs, usually the endoplasmic reticulum of eukaryotes or the periplasm of prokaryotes. It is currently thought to be impossible to produce large amounts of disulfide bond containing protein in the cytoplasm of wild-type bacteria such as E. coli due to the presence of multiple pathways for their reduction.     

Thioredoxin reductase is a key factor in the oxidative stress response of <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> WCFS1

Background  

Thioredoxin (TRX) is a powerful disulfide oxido-reductase that catalyzes a wide spectrum of redox reactions in the cell. The aim of this study is to elucidate the role of the TRX system in the oxidative stress response in Lactobacillus plantarum WCFS1.     

Pre-expression of a sulfhydryl oxidase significantly increases the yields of eukaryotic disulfide bond containing proteins expressed in the cytoplasm of E.coli

Background  

Disulfide bonds are one of the most common post-translational modifications found in proteins. The production of proteins that contain native disulfide bonds is challenging, especially on a large scale. Either the protein needs to be targeted to the endoplasmic reticulum in eukaryotes or to the prokaryotic periplasm. These compartments that are specialised for disulfide bond formation have an active catalyst for their formation, along with catalysts for isomerization to the native state. We have recently shown that it is possible to produce large amounts of prokaryotic disulfide bond containing proteins in the cytoplasm of wild-type bacteria such as E. coli by the introduction of catalysts for both of these processes.     

Stability and assembly in vitro of bacteriophage PP7 virus-like particles

Background  

The stability of a virus-like particle (VLP) is an important consideration for its use in nanobiotechnology. The icosahedral capsid of the RNA bacteriophage PP7 is cross-linked by disulfide bonds between coat protein dimers at its 5-fold and quasi-6-fold symmetry axes. This work determined the effects of these disulfides on the VLP's thermal stability.     

A simplified approach to disulfide connectivity prediction from protein sequences

Background  

Prediction of disulfide bridges from protein sequences is useful for characterizing structural and functional properties of proteins. Several methods based on different machine learning algorithms have been applied to solve this problem and public domain prediction services exist. These methods are however still potentially subject to significant improvements both in terms of prediction accuracy and overall architectural complexity.     

Assembling an arsenal,the scorpion way

Background  

For survival, scorpions depend on a wide array of short neurotoxic polypeptides. The venoms of scorpions from the most studied group, the Buthida, are a rich source of small, 23–78 amino acid-long peptides, well packed by either three or four disulfide bridges that affect ion channel function in excitable and non-excitable cells.     

One-step refolding and purification of disulfide-containing proteins with a C-terminal MESNA thioester

Background  

Expression systems based on self-cleavable intein domains allow the generation of recombinant proteins with a C-terminal thioester. This uniquely reactive C-terminus can be used in native chemical ligation reactions to introduce synthetic groups or to immobilize proteins on surfaces and nanoparticles. Unfortunately, common refolding procedures for recombinant proteins that contain disulfide bonds do not preserve the thioester functionality and therefore novel refolding procedures need to be developed.     

Dissection of an old protein reveals a novel application: domain D of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> Protein A (sSpAD) as a secretion - tag

Background  

Escherichia coli as a frequently utilized host organism for recombinant protein production offers different cellular locations with distinct qualities. The periplasmic space is often favored for the production of complex proteins due to enhanced disulfide bond formation, increased target product stability and simplified downstream processing. To direct proteins to the periplasmic space rather small proteinaceus tags that can be used for affinity purification would be advantageous.     

A Novel Insight into the Oxidoreductase Activity of Helicobacter pylori HP0231 Protein

Background

The formation of a disulfide bond between two cysteine residues stabilizes protein structure. Although we now have a good understanding of the Escherichia coli disulfide formation system, the machineries at work in other bacteria, including pathogens, are poorly characterized. Thus, the objective of this work was to improve our understanding of the disulfide formation machinery of Helicobacter pylori, a leading cause of ulcers and a risk factor for stomach cancer worldwide.

Methods and Results

The protein HP0231 from H. pylori, a structural counterpart of E. coli DsbG, is the focus of this research. Its function was clarified by using a combination of biochemical, microbiological and genetic approaches. In particular, we determined the biochemical properties of HP0231 as well as its redox state in H. pylori cells.

Conclusion

Altogether our results show that HP0231 is an oxidoreductase that catalyzes disulfide bond formation in the periplasm. We propose to call it HpDsbA.     

The Protein Disulfide Isomerase gene family in bread wheat (<Emphasis Type="Italic">T. aestivum L</Emphasis>.)

Background  

The Protein Disulfide Isomerase (PDI) gene family encodes several PDI and PDI-like proteins containing thioredoxin domains and controlling diversified metabolic functions, including disulfide bond formation and isomerisation during protein folding. Genomic, cDNA and promoter sequences of the three homoeologous wheat genes encoding the "typical" PDI had been cloned and characterized in a previous work. The purpose of present research was the cloning and characterization of the complete set of genes encoding PDI and PDI like proteins in bread wheat (Triticum aestivum cv Chinese Spring) and the comparison of their sequence, structure and expression with homologous genes from other plant species.     

Cloning and expression of SOLD1 in ovine and caprine placenta,and their expected roles during the development of placentomes

Background  

The Ly-6 (Ly-6/uPAR) superfamily members share the Ly-6 domain defined by distinct disulfide bonding patterns between 8 or 10 cysteine residues. They comprise membrane- and secretory-type proteins. We recently reported the gene and protein characterization of the bovine secreted protein of Ly-6 domain 1 (SOLD1). Bovine SOLD1 is expressed in trophoblast mononucleate cells (TMCs) and is localized in the cotyledonary mesenchyme. Here, we compared the expression and functionality of SOLD1 among the ruminants. We examined mRNA expression by chorionic fibroblasts as a measure of one of the SOLD1 functions.     

Disulfide Bonds within the C2 Domain of RAGE Play Key Roles in Its Dimerization and Biogenesis

Background

The receptor for advanced glycation end products (RAGE) on the cell surface transmits inflammatory signals. A member of the immunoglobulin superfamily, RAGE possesses the V, C1, and C2 ectodomains that collectively constitute the receptor''s extracellular structure. However, the molecular mechanism of RAGE biogenesis remains unclear, impeding efforts to control RAGE signaling through cellular regulation.

Methodology and Result

We used co-immunoprecipitation and crossing-linking to study RAGE oligomerization and found that RAGE forms dimer-based oligomers. Via non-reducing SDS-polyacrylamide gel electrophoresis and mutagenesis, we found that cysteines 259 and 301 within the C2 domain form intermolecular disulfide bonds. Using a modified tripartite split GFP complementation strategy and confocal microscopy, we also found that RAGE dimerization occurs in the endoplasmic reticulum (ER), and that RAGE mutant molecules without the double disulfide bridges are unstable, and are subjected to the ER-associated degradation.

Conclusion

Disulfide bond-mediated RAGE dimerization in the ER is the critical step of RAGE biogenesis. Without formation of intermolecular disulfide bonds in the C2 region, RAGE fails to reach cell surface.

Significance

This is the first report of RAGE intermolecular disulfide bond.     

Positive genetic interactors of HMG2 identify a new set of genetic perturbations for improving sesquiterpene production in Saccharomyces cerevisiae

Background

Production of correctly disulfide bonded proteins to high yields remains a challenge. Recombinant protein expression in Escherichia coli is the popular choice, especially within the research community. While there is an ever growing demand for new expression strains, few strains are dedicated to post-translational modifications, such as disulfide bond formation. Thus, new protein expression strains must be engineered and the parameters involved in producing disulfide bonded proteins must be understood.

Results

We have engineered a new E. coli protein expression strain named SHuffle, dedicated to producing correctly disulfide bonded active proteins to high yields within its cytoplasm. This strain is based on the trxB gor suppressor strain SMG96 where its cytoplasmic reductive pathways have been diminished, allowing for the formation of disulfide bonds in the cytoplasm. We have further engineered a major improvement by integrating into its chromosome a signal sequenceless disulfide bond isomerase, DsbC. We probed the redox state of DsbC in the oxidizing cytoplasm and evaluated its role in assisting the formation of correctly folded multi-disulfide bonded proteins. We optimized protein expression conditions, varying temperature, induction conditions, strain background and the co-expression of various helper proteins. We found that temperature has the biggest impact on improving yields and that the E. coli B strain background of this strain was superior to the K12 version. We also discovered that auto-expression of substrate target proteins using this strain resulted in higher yields of active pure protein. Finally, we found that co-expression of mutant thioredoxins and PDI homologs improved yields of various substrate proteins.

Conclusions

This work is the first extensive characterization of the trxB gor suppressor strain. The results presented should help researchers design the appropriate protein expression conditions using SHuffle strains.     

Reversible inactivation of yeast mitochondrial phenylalanyl-tRNA synthetase under oxidative stress

Background

Under oxidative stress cytoplasmic aminoacyl-tRNA synthetase (aaRSs) substrate specificity can be compromised, leading to tRNA mischarging and mistranslation of the proteome. Whether similar processes occur in mitochondria, which are major cellular sources of reactive oxygen species (ROS), is unknown. However, relaxed substrate specificity in yeast mitochondrial phenylalanyl-tRNA synthetase (ScmitPheRS) has been reported to increase tRNA mischarging and blocks mitochondrial biogenesis.

The thioredoxin-mediated recycling of Arabidopsis thaliana GRXS16 relies on a conserved C-terminal cysteine

Background

Glutaredoxins (GRXs) are oxidoreductases involved in diverse cellular processes through their capacity to reduce glutathionylated proteins and/or to coordinate iron?sulfur (Fe-S) clusters. Among class II GRXs, the plant-specific GRXS16 is a bimodular protein formed by an N-terminal endonuclease domain fused to a GRX domain containing a ¹⁵⁸CGFS signature.

Conformational characterization of disulfide bonds: A tool for protein classification

Background

Throughout evolution, mutations in particular regions of some protein structures have resulted in extra covalent bonds that increase the overall robustness of the fold: disulfide bonds. The two strategically placed cysteines can also have a more direct role in protein function, either by assisting thiol or disulfide exchange, or through allosteric effects. In this work, we verified how the structural similarities between disulfides can reflect functional and evolutionary relationships between different proteins. We analyzed the conformational patterns of the disulfide bonds in a set of disulfide-rich proteins that included twelve SCOP superfamilies: thioredoxin-like and eleven superfamilies containing small disulfide-rich proteins (SDP).

Results

The twenty conformations considered in the present study were characterized by both structural and energetic parameters. The corresponding frequencies present diverse patterns for the different superfamilies. The least-strained conformations are more abundant for the SDP superfamilies, while the “catalytic” +/−RHook is dominant for the thioredoxin-like superfamily. The “allosteric” -RHSaple is moderately abundant for BBI, Crisp and Thioredoxin-like superfamilies and less frequent for the remaining superfamilies. Using a hierarchical clustering analysis we found that the twelve superfamilies were grouped in biologically significant clusters.

Conclusions

In this work, we carried out an extensive statistical analysis of the conformational motifs for the disulfide bonds present in a set of disulfide-rich proteins. We show that the conformational patterns observed in disulfide bonds are sufficient to group proteins that share both functional and structural patterns and can therefore be used as a criterion for protein classification.     

Disulfide-bonded multimers of proteoglycan 4 (PRG4) are present in normal synovial fluids

Background

The proteoglycan 4 (PRG4) gene encodes for a mucin-like O-linked glycosylated protein with several names, including lubricin and superficial zone protein. The objective of this study was to analyze PRG4 in normal bovine calf and steer synovial fluids for evidence of native multimers formed by intermolecular disulfide bonds.

Methods

A combination of mucin biochemical techniques, with antibodies to both terminal domains and the mucin-like domain of PRG4, were used for analyses.

Results

Multimers were present in both calf and steer fluids, and reduction and alkylation converts the multimeric complex (likely dimeric) into monomeric subunits. Tandem mass spectrometry analyses supported the Western blot data and identified PRG4 in the reduced ∼ 345 kDa monomeric form. Interestingly, ∼ 70 kDa fragments released upon reduction contained peptides from both the N and C terminal regions, which most likely represent fragments of a sparsely glycosylated PRG4 population that are disulfide-linked to extensively glycosylated, intact monomers.

Conclusions

The analyses described here have demonstrated the presence of native disulfide-bonded multimers of PRG4 in normal bovine synovial fluids.

General significance

These structures are similar to those described for multimerization of mucins in general. Such multimerization and proteolytic cleavage of PRG4 may have functional significance in joint health and disease.     

Folding of Matrix Metalloproteinase-2 Prevents Endogenous Generation of MHC Class-I Restricted Epitope

Background

We previously demonstrated that the matrix metalloproteinase-2 (MMP-2) contained an antigenic peptide recognized by a CD8 T cell clone in the HLA-A*0201 context. The presentation of this peptide on class I molecules by human melanoma cells required a cross-presentation mechanism. Surprisingly, the classical endogenous processing pathway did not process this MMP-2 epitope.

Methodology/Principal Findings

By PCR directed mutagenesis we showed that disruption of a single disulfide bond induced MMP-2 epitope presentation. By Pulse-Chase experiment, we demonstrated that disulfide bonds stabilized MMP-2 and impeded its degradation. Finally, using drugs, we documented that mutated MMP-2 epitope presentation used the proteasome and retrotranslocation complex.

Conclusions/Significance

These data appear crucial to us since they established the existence of a new inhibitory mechanism for the generation of a T cell epitope. In spite of MMP-2 classified as a self-antigen, the fact that cross-presentation is the only way to present this MMP-2 epitope underlines the importance to target this type of antigen in immunotherapy protocols.