Disulfide bond formation is probably involved in the biogenesis of approximately one third of human proteins. A central player in this essential process is protein disulfide isomerase or PDI. PDI was the first protein-folding catalyst reported. However, despite more than four decades of study, we still do not understand much about its physiological mechanisms of action. This review examines the published literature with a critical eye. This review aims to (a) provide background on the chemistry of disulfide bond formation and rearrangement, including the concept of reduction potential, before examining the structure of PDI; (b) detail the thiol-disulfide exchange reactions that are catalyzed by PDI in vitro, including a critical examination of the assays used to determine them; (c) examine oxidation and reduction of PDI in vivo, including not only the role of ERo1 but also an extensive assessment of the role of glutathione, as well as other systems, such as peroxide, dehydroascorbate, and a discussion of vitamin K-based systems; (d) consider the in vivo reactions of PDI and the determination and implications of the redox state of PDI in vivo; and (e) discuss other human and yeast PDI-family members.

BackgroundDisulfide 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.ResultsHere we show that the introduction of Erv1p, a sulfhydryl oxidase and a disulfide isomerase allows the efficient formation of natively folded eukaryotic proteins with multiple disulfide bonds in the cytoplasm of E. coli. The production of disulfide bonded proteins was also aided by the use of an appropriate fusion protein to keep the folding intermediates soluble and by choice of media. By combining the pre-expression of a sulfhydryl oxidase and a disulfide isomerase with these other factors, high level expression of even complex disulfide bonded eukaryotic proteins is possibleConclusionsOur results show that the production of eukaryotic proteins with multiple disulfide bonds in the cytoplasm of E. coli is possible. The required exogenous components can be put onto a single plasmid vector allowing facile transfer between different prokaryotic strains. These results open up new avenues for the use of E. coli as a microbial cell factory.

Protein folding in the endoplasmic reticulum is often associated with the formation of native disulfide bonds. Their primary function is to stabilize the folded structure of the protein, although disulfide bond formation can also play a regulatory role. Native disulfide bond formation is not trivial, so it is often the rate‐limiting step of protein folding both in vivo and in vitro. Complex coordinated systems of molecular chaperones and protein folding catalysts have evolved to help proteins attain their correct folded conformation. This includes a family of enzymes involved in catalyzing thiol–disulfide exchange in the endoplasmic reticulum, the protein disulfide isomerase (PDI) family. There are now 17 reported PDI family members in the endoplasmic reticulum of human cells, but the functional differentiation of these is far from complete. Despite PDI being the first catalyst of protein folding reported, there is much that is still not known about its mechanisms of action. This review will focus on the interactions of the human PDI family members with substrates, including recent research on identifying and characterizing their substrate‐binding sites and on determining their natural substrates in vivo.

In bacteria, disulfide bonds confer stability on many proteins exported to the cell envelope or beyond. These proteins include numerous bacterial virulence factors. Thus, bacterial enzymes that promote disulfide bond formation represent targets for compounds inhibiting bacterial virulence. Here, we describe a novel target- and cell-based screening methodology for identifying compounds that inhibit the disulfide bond-forming enzymes E. coli DsbB (EcDsbB) or M. tuberculosis VKOR (MtbVKOR). MtbVKOR can replace EcDsbB although the two are not homologues. Initial screening of 51,487 compounds yielded six specifically inhibiting EcDsbB. These compounds share a structural motif and do not inhibit MtbVKOR. A medicinal chemistry approach led us to select related compounds some of which are much more effective DsbB inhibitors than those found in the screen. These compounds inhibit purified DsbB and prevent anaerobic E. coli growth. Furthermore, these compounds inhibit all but one of the DsbBs of nine other gram-negative pathogenic bacteria tested.

The formation of structural disulfide bonds is essential for the function and stability of a great number of proteins, particularly those that are secreted. There exists a variety of dedicated cellular catalysts and pathways from Archaea to humans that ensure the formation of native disulfide bonds. In this review we describe the initial discoveries of these pathways and report progress in recent years in our understanding of the diversity of these pathways in prokaryotes, including those newly discovered in some Archaea. We will also discuss the various successful efforts to achieve laboratory-based evolution and design of synthetic disulfide bond formation machineries in the bacterium E. coli. These latter studies have also led to new more general insights into the redox environment of the cytoplasm and bacterial cell envelope.