Protein Disulfide Bond Formation in the Periplasm: Determination of the In Vivo Redox State of Cysteine Residues

Denoncin, Katleen; Nicolaes, Valérie; Cho, Seung-Hyun; Leverrier, Pauline; Collet, Jean-François

doi:10.1007/978-1-62703-245-2_20

Cited by 36 publications

(35 citation statements)

References 21 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…As specific anti-CjDsbA1 serum does not exhibit cross reactivity with CjDsbA2 (unpublished data), we used it to determine the CjDsbA1 redox state in vivo in the C. jejuni 81116 wild type strain and in its isogenic dsb mutants, employing the AMS -trapping technique, which distinguishes reduced and oxidized dithiols [38], [39]. The results are presented in Figure 8.…”

Section: Resultsmentioning

confidence: 99%

Functional and Bioinformatics Analysis of Two Campylobacter jejuni Homologs of the Thiol-Disulfide Oxidoreductase, DsbA

et al. 2014

View full text Add to dashboard Cite

BackgroundBacterial Dsb enzymes are involved in the oxidative folding of many proteins, through the formation of disulfide bonds between their cysteine residues. The Dsb protein network has been well characterized in cells of the model microorganism Escherichia coli. To gain insight into the functioning of the Dsb system in epsilon-Proteobacteria, where it plays an important role in the colonization process, we studied two homologs of the main Escherichia coli Dsb oxidase (EcDsbA) that are present in the cells of the enteric pathogen Campylobacter jejuni, the most frequently reported bacterial cause of human enteritis in the world.Methods and ResultsPhylogenetic analysis suggests the horizontal transfer of the epsilon-Proteobacterial DsbAs from a common ancestor to gamma-Proteobacteria, which then gave rise to the DsbL lineage. Phenotype and enzymatic assays suggest that the two C. jejuni DsbAs play different roles in bacterial cells and have divergent substrate spectra. CjDsbA1 is essential for the motility and autoagglutination phenotypes, while CjDsbA2 has no impact on those processes. CjDsbA1 plays a critical role in the oxidative folding that ensures the activity of alkaline phosphatase CjPhoX, whereas CjDsbA2 is crucial for the activity of arylsulfotransferase CjAstA, encoded within the dsbA2-dsbB-astA operon.ConclusionsOur results show that CjDsbA1 is the primary thiol-oxidoreductase affecting life processes associated with bacterial spread and host colonization, as well as ensuring the oxidative folding of particular protein substrates. In contrast, CjDsbA2 activity does not affect the same processes and so far its oxidative folding activity has been demonstrated for one substrate, arylsulfotransferase CjAstA. The results suggest the cooperation between CjDsbA2 and CjDsbB. In the case of the CjDsbA1, this cooperation is not exclusive and there is probably another protein to be identified in C. jejuni cells that acts to re-oxidize CjDsbA1. Altogether the data presented here constitute the considerable insight to the Epsilonproteobacterial Dsb systems, which have been poorly understood so far.

show abstract

Section: Resultsmentioning

confidence: 99%

Functional and Bioinformatics Analysis of Two Campylobacter jejuni Homologs of the Thiol-Disulfide Oxidoreductase, DsbA

et al. 2014

View full text Add to dashboard Cite

show abstract

“…The fractions of reduced and oxidized PaDsbA2 were determined using 4-acetamido-4′-maleimidylstilbene-2,2′-disulfonic acid (AMS) trapping by the method of Denoncin et al (57). Briefly, PaDsbA2 and PaDsbA2 CCSS (1 µM) were incubated overnight at room temperature in 50 mM KPi pH 7.0, 0.1 mM EDTA and various glutathione (GSH)/glutathione disulfide (GSSG) ratios.…”

Section: Methodsmentioning

confidence: 99%

Dissecting the Machinery That Introduces Disulfide Bonds in Pseudomonas aeruginosa

Arts

Ball

Leverrier

et al. 2013

mBio

Self Cite

View full text Add to dashboard Cite

Disulfide bond formation is required for the folding of many bacterial virulence factors. However, whereas the Escherichia coli disulfide bond-forming system is well characterized, not much is known on the pathways that oxidatively fold proteins in pathogenic bacteria. Here, we report the detailed unraveling of the pathway that introduces disulfide bonds in the periplasm of the human pathogen Pseudomonas aeruginosa. The genome of P. aeruginosa uniquely encodes two DsbA proteins (P. aeruginosa DsbA1 [PaDsbA1] and PaDsbA2) and two DsbB proteins (PaDsbB1 and PaDsbB2). We found that PaDsbA1, the primary donor of disulfide bonds to secreted proteins, is maintained oxidized in vivo by both PaDsbB1 and PaDsbB2. In vitro reconstitution of the pathway confirms that both PaDsbB1 and PaDsbB2 shuttle electrons from PaDsbA1 to membrane-bound quinones. Accordingly, deletion of both P. aeruginosa dsbB1 (PadsbB1) and PadsbB2 is required to prevent the folding of several P. aeruginosa virulence factors and to lead to a significant decrease in pathogenicity. Using a high-throughput proteomic approach, we also analyzed the impact of PadsbA1 deletion on the global periplasmic proteome of P. aeruginosa, which allowed us to identify more than 20 new potential substrates of this major oxidoreductase. Finally, we report the biochemical and structural characterization of PaDsbA2, a highly oxidizing oxidoreductase, which seems to be expressed under specific conditions. By fully dissecting the machinery that introduces disulfide bonds in P. aeruginosa, our work opens the way to the design of novel antibacterial molecules able to disarm this pathogen by preventing the proper assembly of its arsenal of virulence factors.

show abstract

“…The thiol modifying agent 4-acetamido-4¢-maleimidylstilbene-2,2¢-disulfonic acid (AMS) reacts with free cysteines in the protein increasing its molecular weight in 540 Da per AMS molecule added (8).…”

Section: Recombinant Fura Contains Two Intramolecular Disulfide Bridgmentioning

confidence: 99%

Cysteine Mutational Studies Provide Insight into a Thiol-Based Redox Switch Mechanism of Metal and DNA Binding in FurA fromAnabaenasp. PCC 7120

Botello-Morte

Pellicer

Sein‐Echaluce

et al. 2016

Antioxidants & Redox Signaling

View full text Add to dashboard Cite

Aims: The ferric uptake regulator (Fur) is the main transcriptional regulator of genes involved in iron homeostasis in most prokaryotes. FurA from Anabaena sp. PCC 7120 contains five cysteine residues, four of them arranged in two redox-active CXXC motifs. The protein needs not only metal but also reducing conditions to remain fully active in vitro. Through a mutational study of the cysteine residues present in FurA, we have investigated their involvement in metal and DNA binding. , suggesting that the environments of these cysteines are mutually interdependent. Innovation: We propose that C 101 is part of a thiol/disulfide redox switch that determines FurA ability to bind the metal corepressor. Conclusion: This mechanism supports a novel feature of a Fur protein that emerges as a regulator, which connects the response to changes in the intracellular redox state and iron management in cyanobacteria. Antioxid. Redox Signal. 24, 173-185.

show abstract

Protein Disulfide Bond Formation in the Periplasm: Determination of the In Vivo Redox State of Cysteine Residues

Cited by 36 publications

References 21 publications

Functional and Bioinformatics Analysis of Two Campylobacter jejuni Homologs of the Thiol-Disulfide Oxidoreductase, DsbA

Functional and Bioinformatics Analysis of Two Campylobacter jejuni Homologs of the Thiol-Disulfide Oxidoreductase, DsbA

Dissecting the Machinery That Introduces Disulfide Bonds in Pseudomonas aeruginosa

Cysteine Mutational Studies Provide Insight into a Thiol-Based Redox Switch Mechanism of Metal and DNA Binding in FurA fromAnabaenasp. PCC 7120

Contact Info

Product

Resources

About