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Disulfide bond formation

The formation of disulfide bonds in proteins is an oxidative process that generates a covalent bond linking the sulfur atoms of two cysteine residues. Disulfide bond formation has a central role in protein folding of both eukaryotes and prokaryotes.

Disulfide-bonded proteins are located in more oxidizing environments such as the bacterial cell envelope. In Escherichia coli the enzymes involved in disulfide bond formation are the periplasmic thioredoxin homologue DsbA, which introduces disulfide bonds into substrate proteins, and the membrane protein DsbB, which maintains DsbA active by oxidizing it.

three pathways of disulfide bond formation

Most Proteobacteria (like Pseudomonas aeruginosa) use DsbA- and DsbB-like proteins to form disulfide bonds in exported proteins. However, Actinobacteria (like Mycobacterium tuberculosis), Cyanobacteria and aerobic δ-proteobacteria maintain DsbA oxidized not by DsbB, but by the protein VKOR, a homologue of human Vitamin K epoxide reductase.

In bacteria, disulfide bonds play a role in the folding and stability of proteins involved in important cellular processes such as cell division, assembly of the outer membrane and virulence of gram-negative bacteria. Therefore, the pathways that introduce disulfide bonds into proteins represent good targets for the development of new antibacterial therapies.

The goal of our research is to study the fundamental biology of disulfide bond formation in bacteria as well as to find inhibitors of this process that is crucial for growth and virulence of pathogenic bacteria for which new antibacterial agents are most desperately needed.

We have developed a method to look for molecules that could be used as antibiotics, antivirulence therapeutics or anticoagulants using E. coli cells. After several rounds of screening of several synthetic small molecule libraries we have found few candidate inhibitors of the DsbB and VKOR enzymes of pathogenic bacteria.

We are currently working on one family of inhibitors that resemble anticoagulants in order to find more effective inhibitors of bacterial VKOR . We are also using these molecules to understand more about the catalytic mechanisms of these type of enzymes.

We are interested in expanding our inhibitor search using natural products libraries.

We aim to enable the systematic identification of substrates of the disulfide bond formation pathway of human pathogens.

We have identified some of the first substrates of DsbA in mycobacteria and we are currently developing a proteomics method to trap substrates attached to DsbA in M. smegmatis.

We aim to determine the selectivity and substrate preference of DsbA homologues in Mycobacteria and E. coli by swapping their DsbA proteins.

We are performing genetic screens to find the synthetic lethal genes of disulfide bond forming systems in E. coli and P. aeruginosa.

We will study synthetic lethal genes that can participate in redox reactions in both model organisms.

We aim to understand how anaerobic organisms catalyze disulfide bond formation. We have identified DsbA and VKOR homologues in Clostridial species and we are interested in understanding what electron aceptors are capable of sustaining oxidative protein folding.

We have also identified VKOR homologues in unusual organisms. We aim to expand the repertoire of functions that these enzymes sustain in all domains of life.

Research

Disulfide bond formation

Antimicrobial screenings

Understanding DsbA diversity and selectivity

Essentiality of disulfide bond-forming proteins

Diversity of disulfide bond formation systems

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