Brussels Center for Redox Biology

Brussels, Belgium

Brussels Center for Redox Biology

Brussels, Belgium

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Roos G.,Vrije Universiteit Brussel | Roos G.,Brussels Center for Redox Biology | Foloppe N.,51 Natal Road | Messens J.,Vlaams Instituut voor Biotechnologie VIB | And 2 more authors.
Antioxidants and Redox Signaling | Year: 2013

Many cellular functions involve cysteine chemistry via thiol-disulfide exchange pathways. The nucleophilic cysteines of the enzymes involved are activated as thiolate. A thiolate is much more reactive than a neutral thiol. Therefore, determining and understanding the pKas of functional cysteines are important aspects of biochemistry and molecular biology with direct implications for redox signaling. Here, we describe the experimental and theoretical methods to determine cysteine pKa values, and we examine the factors that control these pKas. Drawing largely on experience gained with the thioredoxin superfamily, we examine the roles of solvation, charge-charge, helix macrodipole, and hydrogen bonding interactions as pK a-modulating factors. The contributions of these factors in influencing cysteine pKas and the associated chemistry, including the relevance for the reaction kinetics and thermodynamics, are discussed. This analysis highlights the critical role of direct hydrogen bonding to the cysteine sulfur as a key factor modulating the equilibrium between thiol S-H and thiolate S-. This role is easily understood intuitively and provides a framework for biochemical functional insights. © 2013, Mary Ann Liebert, Inc.


Van Laer K.,Vlaams Instituut voor Biotechnologie VIB | Van Laer K.,Vrije Universiteit Brussel | Van Laer K.,Brussels Center for Redox Biology | Hamilton C.J.,University of East Anglia | And 3 more authors.
Antioxidants and Redox Signaling | Year: 2013

Significance: Oxidative stress is widely invoked in inflammation, aging, and complex diseases. To avoid unwanted oxidations, the redox environment of cellular compartments needs to be tightly controlled. The complementary action of oxidoreductases and of high concentrations of low-molecular-weight (LMW) nonprotein thiols plays an essential role in maintaining the redox potential of the cell in balance. Recent Advances: While LMW thiols are central players in an extensive range of redox regulation/metabolism processes, not all organisms use the same thiol cofactors to this effect, as evidenced by the recent discovery of mycothiol (MSH) and bacillithiol (BSH) among different gram-positive bacteria. Critical Issues: LMW thiol-disulfide exchange processes and their cellular implications are often oversimplified, as only the biology of the free thiols and their symmetrical disulfides is considered. In bacteria under oxidative stress, especially where concentrations of different LMW thiols are comparable [e.g., BSH, coenzyme A (CoA), and cysteine (Cys) in many low-G+C gram-positive bacteria (Firmicutes)], mixed disulfides (e.g., CoASSB and CySSCoA) must surely be major thiol-redox metabolites that need to be taken into consideration. Future Directions: There are many microorganisms whose LMW thiol-redox buffers have not yet been identified (either bioinformatically or experimentally). Many elements of BSH and MSH redox biochemistry remain to be explored. The fundamental biophysical properties, thiol pKa and redox potential, have not yet been determined, and the protein interactome in which the biothiols MSH and BSH are involved needs further exploration. © 2013 Mary Ann Liebert, Inc.


Olah J.,Budapest University of Technology and Economics | Van Bergen L.,Vrije Universiteit Brussel | De Proft F.,Vrije Universiteit Brussel | Roos G.,Vrije Universiteit Brussel | Roos G.,Brussels Center for Redox Biology
Journal of Biomolecular Structure and Dynamics | Year: 2015

Protein thiol/sulfenic acid oxidation potentials provide a tool to select specific oxidation agents, but are experimentally difficult to obtain. Here, insights into the thiol sulfenylation thermodynamics are obtained from model calculations on small systems and from a quantum mechanics/molecular mechanics (QM/MM) analysis on human 2-Cys peroxiredoxin thioredoxin peroxidase B (Tpx-B). To study thiol sulfenylation in Tpx-B, our recently developed computational method to determine reduction potentials relatively compared to a reference system and based on reaction energies reduction potential from electronic energies is updated. Tpx-B forms a sulfenic acid (R-SO-) on one of its active site cysteines during reactive oxygen scavenging. The observed effect of the conserved active site residues is consistent with the observed hydrogen bond interactions in the QM/MM optimized Tpx-B structures and with free energy calculations on small model systems. The ligand effect could be linked to the complexation energies of ligand L with CH3S- and CH3SO-. Compared to QM only calculations on Tpx-Bs active site, the QM/MM calculations give an improved understanding of sulfenylation thermodynamics by showing that other residues from the protein environment other than the active site residues can play an important role. © 2014 Taylor & Francis.


Denoncin K.,Catholic University of Louvain | Denoncin K.,Brussels Center for Redox Biology | Vertommen D.,Catholic University of Louvain | Paek E.,University of Seoul | And 2 more authors.
Journal of Biological Chemistry | Year: 2010

The assembly of the β-barrel proteins present in the outer membrane (OM) of Gram-negative bacteria is poorly characterized. After translocation across the inner membrane, unfolded β-barrel proteins are escorted across the periplasm by chaperones that reside within this compartment. Two partially redundant chaperones, SurA and Skp, are considered to transport the bulk mass of β-barrel proteins. We found that the periplasmic disulfide isomerase DsbC cooperates with SurA and the thiol oxidase DsbA in the folding of the essential β-barrel protein LptD. LptD inserts lipopolysaccharides in the OM. It is also the only β-barrel protein with more than two cysteine residues. We found that surAdsbC mutants, but not skpdsbC mutants, exhibit a synthetic phenotype. They have a decreased OM integrity, which is due to the lack of the isomerase activity of DsbC. We also isolated DsbC in a mixed disulfide complex with LptD. As such, LptD is identified as the first substrate of DsbC that is localized in the OM. Thus, electrons flowing from the cytoplasmic thioredoxin system maintain the integrity of the OM by assisting the folding of one of the most important β-barrel proteins. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.


Roos G.,Vlaams Institute for Biotechnology | Roos G.,Vrije Universiteit Brussel | Roos G.,Brussels Center for Redox Biology | Messens J.,Vlaams Institute for Biotechnology | And 2 more authors.
Free Radical Biology and Medicine | Year: 2011

Protein sulfenic acid formation has long been regarded as unwanted damage caused by reactive oxygen species (ROS). However, over the past 10 years, accumulating evidence has shown that the reversible oxidation of cysteine thiol groups to sulfenic acid functions as a redox-based signal transduction mechanism. Here, we review the mechanisms of sulfenic acid formation by ROS. We present some of the most important roles played by sulfenic acids in living cells as well as the pathways that regulate sulfenic acid formation. We highlight the experimental tools that have been developed to study the cellular sulfenome and show how computational approaches might help to better understand the mechanisms of sulfenic acid formation. © 2011 Elsevier Inc.


Roszczenko P.,University of Warsaw | Radomska K.A.,University of Warsaw | Wywial E.,International Institute of Molecular and Cell Biology | Collet J.-F.,Catholic University of Louvain | And 2 more authors.
PLoS ONE | Year: 2012

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. © 2012 Roszczenko et al.


Messens J.,Brussels Center for Redox Biology | Messens J.,Vrije Universiteit Brussel | Collet J.-F.,Brussels Center for Redox Biology | Collet J.-F.,Catholic University of Leuven
Antioxidants and Redox Signaling | Year: 2013

The major function of disulfide bonds is not only the stabilization of protein structures. Over the last 30 years, a change in perspective took place driven by groundbreaking experiments, which promoted disulfide bonds to central players in essential thiol-disulfide exchange reactions involved in signal transduction, thiol protection, and redox homeostasis regulation. This new view stimulated redox research and led to the discovery of novel redox pathways, redox enzymes, and new low-molecular-weight thiols. These redox-sensitive molecules operate along diverse pathways via a dynamic thiol-disulfide mechanism in which disulfide bonds are reversibly formed and reduced, thereby switching the molecules between different conformational and functional states. It is now clear that disulfide bonds play a pivotal role in cellular reduction and oxidation processes. However, in spite of the fundamental cell biological and medical importance of the thiol-disulfide exchange switches, we are only beginning to understand their principles of specificity, their mechanism of action, and their role in signal transduction. Our further progress in understanding the thiol-disulfide switches will strongly depend on the chemical tools and on the technological advances that will be made in the development of new methodologies. Antioxid. Redox Signal. 18, 1594-1596. © Copyright 2013, Mary Ann Liebert, Inc. 2013. © 2013 Mary Ann Liebert, Inc.


Collet J.-F.,Catholic University of Leuven | Collet J.-F.,Brussels Center for Redox Biology | Messens J.,Brussels Center for Redox Biology | Messens J.,Vrije Universiteit Brussel
Antioxidants and Redox Signaling | Year: 2010

Thioredoxins are ubiquitous antioxidant enzymes that play important roles in many health-related cellular processes. As such, the fundamental knowledge of how these enzymes work is of prime importance for understanding cellular redox mechanisms and for laying the ground for the development of future therapeutic approaches. Over the past 40 years, a really impressive amount of data has been published on thioredoxins. Here, we review the most significant results that have contributed to our knowledge regarding the structure, the function, and the mechanism of these crucial enzymes. © 2010, Mary Ann Liebert, Inc.


Billiet L.,Vrije Universiteit Brussel | Billiet L.,Brussels Center for Redox Biology | Geerlings P.,Vrije Universiteit Brussel | Messens J.,Vrije Universiteit Brussel | And 3 more authors.
Free Radical Biology and Medicine | Year: 2012

Protein sulfenic acids are essential cysteine oxidations in cellular signaling pathways. The thermodynamics that drive protein sulfenylation are not entirely clear. Experimentally, sulfenic acid reduction potentials are hard to measure, because of their highly reactive nature. We designed a calculation method, the reduction potentials from electronic energies (REE) method, to give for the first time insight into the thermodynamic aspects of protein sulfenylation. The REE method is based on the correlation between reaction path-independent reaction energies and free energies of a series of analogous reactions. For human peroxiredoxin (Tpx-B), an antioxidant enzyme that forms a sulfenic acid on one of its active-site cysteines during reactive oxygen scavenging, we found that the reduction potential depends on the composition of the active site and on the protonation state of the cysteine. Interaction with polar residues directs the RSO -/RS - reduction to a lower potential than the RSOH/RSH reduction. A conserved arginine that thermodynamically favors the sulfenylation reaction might be a good candidate to favor the reaction kinetics. The REE method is not limited to thiol sulfenylation, but can be broadly applied to understand protein redox biology in general. © 2012 Elsevier Inc. All rights reserved.


Van Laer K.,Vrije Universiteit Brussel | Van Laer K.,Brussels Center for Redox Biology | Oliveira M.,Vrije Universiteit Brussel | Oliveira M.,Brussels Center for Redox Biology | And 4 more authors.
Protein Science | Year: 2014

NrdH-redoxins shuffle electrons from the NADPH pool in the cell to Class Ib ribonucleotide reductases, which in turn provide the precursors for DNA replication and repair. NrdH-redoxins have a CVQC active site motif and belong to the thioredoxin-fold protein family. As for other thioredoxinfold proteins, the pKa of the nucleophilic cysteine of NrdH-redoxins is of particular interest since it affects the catalytic reaction rate of the enzymes. Recently, the pKa value of this cysteine in Corynebacterium glutamicum and Mycobacterium tuberculosis NrdH-redoxins were determined, but structural insights explaining the relatively low pKa remained elusive. We subjected C. glutamicum NrdHredoxin to an extensive molecular dynamics simulation to expose the factors regulating the pKa of the nucleophilic cysteine. We found that the nucleophilic cysteine receives three hydrogen bonds from residues within the CVQC active site motif. Additionally, a fourth hydrogen bond with a lysine located N-terminal of the active site further lowers the cysteine pKa. However, site-directed mutagenesis data show that the major contribution to the lowering of the cysteine pKa comes from the positive charge of the lysine and not from the additional Lys-Cys hydrogen bond. In 12% of the NrdHredoxin family, this lysine is replaced by an arginine that also lowers the cysteine pKa. All together, the four hydrogen bonds and the electrostatic effect of a lysine or an arginine located N-terminally of the active site dynamically regulate the pKa of the nucleophilic cysteine in NrdH-redoxins. © 2013 The Protein Society.

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