scholarly journals Modulation of Thiol-Disulfide Oxidoreductases for Increased Production of Disulfide-Bond-Containing Proteins in Bacillus subtilis

2008 ◽  
Vol 74 (24) ◽  
pp. 7536-7545 ◽  
Author(s):  
Thijs R. H. M. Kouwen ◽  
Jean-Yves F. Dubois ◽  
Roland Freudl ◽  
Wim J. Quax ◽  
Jan Maarten van Dijl
Keyword(s):  
Bacillus Subtilis ◽  
Disulfide Bond ◽  
Disulfide Bonds ◽  
Structural Integrity ◽  
Downstream Processing ◽  
Cell Factory ◽  
Biotechnological Production ◽  
Redox Active ◽  
Model Protein

ABSTRACT Disulfide bonds are important for the correct folding, structural integrity, and activity of many biotechnologically relevant proteins. For synthesis and subsequent secretion of these proteins in bacteria, such as the well-known “cell factory” Bacillus subtilis, it is often the correct formation of disulfide bonds that is the greatest bottleneck. Degradation of inefficiently or incorrectly oxidized proteins and the requirement for costly and time-consuming reduction and oxidation steps in the downstream processing of the proteins still are major limitations for full exploitation of B. subtilis for biopharmaceutical production. Therefore, the present study was aimed at developing a novel in vivo strategy for improved production of secreted disulfide-bond-containing proteins. Three approaches were tested: depletion of the major cytoplasmic reductase TrxA; introduction of the heterologous oxidase DsbA from Staphylococcus carnosus; and addition of redox-active compounds to the growth medium. As shown using the disulfide-bond-containing molecule Escherichia coli PhoA as a model protein, combined use of these three approaches resulted in secretion of amounts of active PhoA that were ∼3.5-fold larger than the amounts secreted by the parental strain B. subtilis 168. Our findings indicate that Bacillus strains with improved oxidizing properties can be engineered for biotechnological production of heterologous high-value proteins containing disulfide bonds.

scholarly journals A Disulfide Bond-Containing Alkaline Phosphatase Triggers a BdbC-Dependent Secretion Stress Response in Bacillus subtilis

2006 ◽  
Vol 72 (11) ◽  
pp. 6876-6885 ◽  
Author(s):  
Elise Darmon ◽  
Ronald Dorenbos ◽  
Jochen Meens ◽  
Roland Freudl ◽  
Haike Antelmann ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Stress Response ◽  
Disulfide Bond ◽  
Disulfide Bonds ◽  
Protein Quality ◽  
Secretory Protein ◽  
Secretory Proteins ◽  
Biotechnological Production ◽  
Secretion Stress ◽  
High Level

ABSTRACT The gram-positive bacterium Bacillus subtilis secretes high levels of proteins into its environment. Most of these secretory proteins are exported from the cytoplasm in an unfolded state and have to fold efficiently after membrane translocation. As previously shown for α-amylases of Bacillus species, inefficient posttranslocational protein folding is potentially detrimental and stressful. In B. subtilis, this so-called secretion stress is sensed and combated by the CssRS two-component system. Two known members of the CssRS regulon are the htrA and htrB genes, encoding potential extracytoplasmic chaperone proteases for protein quality control. In the present study, we investigated whether high-level production of a secretory protein with two disulfide bonds, PhoA of Escherichia coli, induces secretion stress in B. subtilis. Our results show that E. coli PhoA production triggers a relatively moderate CssRS-dependent secretion stress response in B. subtilis. The intensity of this response is significantly increased in the absence of BdbC, which is a major determinant for posttranslocational folding of disulfide bond-containing proteins in B. subtilis. Our findings show that BdbC is required to limit the PhoA-induced secretion stress. This conclusion focuses interest on the BdbC-dependent folding pathway for biotechnological production of proteins with disulfide bonds in B. subtilis and related bacilli.

scholarly journals Disulfide Transfer between Two Conserved Cysteine Pairs Imparts Selectivity to Protein Oxidation by Ero1

2006 ◽  
Vol 17 (5) ◽  
pp. 2256-2266 ◽  
Author(s):  
Carolyn S. Sevier ◽  
Chris A. Kaiser
Keyword(s):  
Disulfide Bond ◽  
Oxidase Activity ◽  
Disulfide Bonds ◽  
Structural Changes ◽  
Catalytic Mechanism ◽  
Structural Data ◽  
Secretory Proteins ◽  
Mixed Disulfide ◽  
Redox Active

The membrane-associated flavoprotein Ero1p promotes disulfide bond formation in the endoplasmic reticulum (ER) by selectively oxidizing the soluble oxidoreductase protein disulfide isomerase (Pdi1p), which in turn can directly oxidize secretory proteins. Two redox-active disulfide bonds are essential for Ero1p oxidase activity: Cys100-Cys105 and Cys352-Cys355. Genetic and structural data indicate a disulfide bond is transferred from Cys100-Cys105 directly to Pdi1p, whereas a Cys352-Cys355 disulfide bond is used to reoxidize the reduced Cys100-Cys105 pair through an internal thiol-transfer reaction. Electron transfer from Cys352-Cys355 to molecular oxygen, by way of a flavin cofactor, maintains Cys352-Cys355 in an oxidized form. Herein, we identify a mixed disulfide species that confirms the Ero1p intercysteine thiol-transfer relay in vivo and identify Cys105 and Cys352 as the cysteines that mediate thiol-disulfide exchange. Moreover, we describe Ero1p mutants that have the surprising ability to oxidize substrates in the absence of Cys100-Cys105. We show the oxidase activity of these mutants results from structural changes in Ero1p that allow substrates increased access to Cys352-Cys355, which are normally buried beneath the protein surface. The altered activity of these Ero1p mutants toward selected substrates leads us to propose the catalytic mechanism involving transfer between cysteine pairs evolved to impart substrate specificity to Ero1p.

Galectin-13/placental protein 13: redox-active disulfides as switches for regulating structure, function and cellular distribution

Glycobiology ◽  
2019 ◽  
Vol 30 (2) ◽  
pp. 120-129 ◽  
Author(s):  
Tong Yang ◽  
Yuan Yao ◽  
Xing Wang ◽  
Yuying Li ◽  
Yunlong Si ◽  
...  
Keyword(s):  
Disulfide Bond ◽  
Disulfide Bonds ◽  
Bond Formation ◽  
Gel Filtration ◽  
Wild Type ◽  
High Concentration ◽  
Redox Active ◽  
Placental Protein ◽  
The Relationship ◽  
Low Expression

Abstract Galectin-13 (Gal-13) plays numerous roles in regulating the relationship between maternal and fetal tissues. Low expression levels or mutations of the lectin can result in pre-eclampsia. The previous crystal structure and gel filtration data show that Gal-13 dimerizes via formation of two disulfide bonds formed by Cys136 and Cys138. In the present study, we mutated them to serine (C136S, C138S and C136S/C138S), crystalized the variants and solved their crystal structures. All variants crystallized as monomers. In the C136S structure, Cys138 formed a disulfide bond with Cys19, indicating that Cys19 is important for regulation of reversible disulfide bond formation in this lectin. Hemagglutination assays demonstrated that all variants are inactive at inducing erythrocyte agglutination, even though gel filtration profiles indicate that C136S and C138S could still form dimers, suggesting that these dimers do not exhibit the same activity as wild-type (WT) Gal-13. In HeLa cells, the three variants were found to be distributed the same as with WT Gal-13. However, a Gal-13 variant (delT221) truncated at T221 could not be transported into the nucleus, possibly explaining why women having this variant get pre-eclampsia. Considering the normally high concentration of glutathione in cells, WT Gal-13 should exist mostly as a monomer in cytoplasm, consistent with the monomeric variant C136S/C138S, which has a similar ability to interact with HOXA1 as WT Gal-13.

scholarly journals Evolution Rescues Folding of Human Immunodeficiency Virus-1 Envelope Glycoprotein GP120 Lacking a Conserved Disulfide Bond

2008 ◽  
Vol 19 (11) ◽  
pp. 4707-4716 ◽  
Author(s):  
Rogier W. Sanders ◽  
Shang-Te D. Hsu ◽  
Eelco van Anken ◽  
I. Marije Liscaljet ◽  
Martijn Dankers ◽  
...  
Keyword(s):  
Disulfide Bond ◽  
Disulfide Bonds ◽  
Side Chain ◽  
Viral Glycoprotein ◽  
Immunodeficiency Virus ◽  
Glycoprotein Gp120 ◽  
Dynamics Simulations ◽  
Β Sheet

The majority of eukaryotic secretory and membrane proteins contain disulfide bonds, which are strongly conserved within protein families because of their crucial role in folding or function. The exact role of these disulfide bonds during folding is unclear. Using virus-driven evolution we generated a viral glycoprotein variant, which is functional despite the lack of an absolutely conserved disulfide bond that links two antiparallel β-strands in a six-stranded β-barrel. Molecular dynamics simulations revealed that improved hydrogen bonding and side chain packing led to stabilization of the β-barrel fold, implying that β-sheet preference codirects glycoprotein folding in vivo. Our results show that the interactions between two β-strands that are important for the formation and/or integrity of the β-barrel can be supported by either a disulfide bond or β-sheet favoring residues.

scholarly journals Characterization of Two Homologous Disulfide Bond Systems Involved in Virulence Factor Biogenesis in Uropathogenic Escherichia coli CFT073

2009 ◽  
Vol 191 (12) ◽  
pp. 3901-3908 ◽  
Author(s):  
Makrina Totsika ◽  
Begoña Heras ◽  
Daniël J. Wurpel ◽  
Mark A. Schembri
Keyword(s):  
Escherichia Coli ◽  
Disulfide Bond ◽  
Disulfide Bonds ◽  
Infection Model ◽  
Multicopy Plasmid ◽  
Unfolded Proteins ◽  
Upec Strain ◽  
Arylsulfate Sulfotransferase ◽  
Redox Active ◽  
Strain Cft073

ABSTRACT Disulfide bond (DSB) formation is catalyzed by disulfide bond proteins and is critical for the proper folding and functioning of secreted and membrane-associated bacterial proteins. Uropathogenic Escherichia coli (UPEC) strains possess two paralogous disulfide bond systems: the well-characterized DsbAB system and the recently described DsbLI system. In the DsbAB system, the highly oxidizing DsbA protein introduces disulfide bonds into unfolded polypeptides by donating its redox-active disulfide and is in turn reoxidized by DsbB. DsbA has broad substrate specificity and reacts readily with reduced unfolded proteins entering the periplasm. The DsbLI system also comprises a functional redox pair; however, DsbL catalyzes the specific oxidative folding of the large periplasmic enzyme arylsulfate sulfotransferase (ASST). In this study, we characterized the DsbLI system of the prototypic UPEC strain CFT073 and examined the contributions of the DsbAB and DsbLI systems to the production of functional flagella as well as type 1 and P fimbriae. The DsbLI system was able to catalyze disulfide bond formation in several well-defined DsbA targets when provided in trans on a multicopy plasmid. In a mouse urinary tract infection model, the isogenic dsbAB deletion mutant of CFT073 was severely attenuated, while deletion of dsbLI or assT did not affect colonization.

scholarly journals On the Functional Interchangeability, Oxidant versus Reductant, of Members of the Thioredoxin Superfamily

2000 ◽  
Vol 182 (3) ◽  
pp. 723-727 ◽  
Author(s):  
Laurent Debarbieux ◽  
Jon Beckwith
Keyword(s):  
Disulfide Bond ◽  
Disulfide Bonds ◽  
Signal Sequence ◽  
Cell Envelope ◽  
Bond Formation ◽  
Disulfide Bond Formation ◽  
Thioredoxin 1 ◽  
High Level

ABSTRACT Escherichia coli thioredoxin 1 has been characterized in vivo and in vitro as one of the most efficient reductants of disulfide bonds. Nevertheless, under some conditions, thioredoxin 1 can also act in vivo as an oxidant, promoting formation of disulfide bonds in the cytoplasm (E. J. Stewart, F. Åslund, and J. Beckwith, EMBO J. 17:5543–5550, 1998). We recently showed that when a signal sequence is attached to thioredoxin 1 it is exported to the periplasm, where it can also act as an oxidant, replacing the normal periplasmic catalyst of disulfide bond formation, DsbA, in oxidizing cell envelope proteins (L. Debarbieux and J. Beckwith, Proc. Natl. Acad. Sci. USA 95:10751–10756, 1998). Here we report pulse-chase studies of the efficiency of disulfide bond formation in strains exporting thioredoxin 1 and more-oxidizing variants of it. While the exported thioredoxin 1 itself substantially speeds up the kinetics of disulfide bond formation, a version of this protein containing the DsbA active site exhibits kinetics that are indistinguishable from those of the DsbA protein itself. Further, we confirm the findings of Jonda et al. (S. Jonda, M. Huber-Wunderlich, R. Glockshuber, and E. Mössner, EMBO J. 18:3271–3281, 1999), who found that DsbB is responsible for the oxidation of exported thioredoxin 1, and we report the detection of a disulfide-bonded DsbB-thioredoxin 1 complex. Finally, we have found that under conditions of high-level expression of exported thioredoxin 1, the protein can act as both an oxidant and a reductant.

scholarly journals Characterization of the SARS-CoV-2 coronavirus X4-like accessory protein

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Olanrewaju Ayodeji Durojaye ◽  
Nkwachukwu Oziamara Okoro ◽  
Arome Solomon Odiba
Keyword(s):  
Rapid Development ◽  
Protein A ◽  
The Novel ◽  
Quality Model ◽  
A Value ◽  
Model Protein ◽  
Novel Coronavirus ◽  
Global Threat

Abstract Background The novel coronavirus SARS-CoV-2 is currently a global threat to health and economies. Therapeutics and vaccines are in rapid development; however, none of these therapeutics are considered as absolute cure, and the potential to mutate makes it necessary to find therapeutics that target a highly conserved regions of the viral structure. Results In this study, we characterized an essential but poorly understood coronavirus accessory X4 protein, a core and stable component of the SARS-CoV family. Sequence analysis shows a conserved ~ 90% identity between the SARS-CoV-2 and previously characterized X4 protein in the database. QMEAN Z score of the model protein shows a value of around 0.5, within the acceptable range 0–1. A MolProbity score of 2.96 was obtained for the model protein and indicates a good quality model. The model has Ramachandran values of φ = − 57o and ψ = − 47o for α-helices and values of φ = − 130o and ψ = + 140o for twisted sheets. Conclusions The protein data obtained from this study provides robust information for further in vitro and in vivo experiment, targeted at devising therapeutics against the virus. Phylogenetic analysis further supports previous evidence that the SARS-CoV-2 is positioned with the SL-CoVZC45, BtRs-BetaCoV/YN2018B and the RS4231 Bat SARS-like corona viruses.

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